Immobilized lipase using grafted modified amino resin as carrier and preparation method thereof

Abstract

The present invention relates to the field of immobilized enzymes, and in particular to an immobilized lipase using a grafted modified amino resin as a carrier and a preparation method thereof. The method first uses the oxidative self-polymerization property of tannic acid to modify the amino resin to obtain a first modified amino resin; and uses a fatty amine as a hydrophobic modifier to modify the first modified amino resin to obtain a second modified amino resin. Subsequently, the lipase is immobilized on the second modified amino resin by physical adsorption, and the lipase undergoes interfacial activation at the amphiphilic interface on the second modified amino resin. A portion of the lipase is covalently bound to the quinone group in the activated state, thereby obtaining an immobilized lipase with high activity and stability. The present invention is simple to operate, green and non-toxic, and the immobilized lipase prepared has excellent enzyme activity and stability. It is a method for preparing immobilized lipase on a large scale, can be applied to the food field, and has the potential for industrial application.

Description

Technical Field

[0001] The present invention relates to the field of immobilized enzymes, in particular to an immobilized lipase using a grafted modified amino resin as a carrier and a preparation method thereof. Background Art

[0002] Lipase (EC3.1.1.3), a green and efficient biocatalyst, has wide applications in biodiesel production, chiral pharmaceuticals, and the preparation of functional oils. However, free lipase is highly sensitive to the operating environment and easily inactivated by factors such as temperature, pH, and organic solvents. Furthermore, it is difficult to recycle, severely limiting its industrial application. Enzyme immobilization techniques, which immobilize enzymes onto supports through physical adsorption, covalent bonding, crosslinking, and encapsulation, can significantly improve enzyme stability and reusability. As a typical interface-activated enzyme, lipase's active center is located in a hydrophobic "pocket" covered by a "lid" structure that is hydrophilic on the outside and hydrophobic on the inside. When lipase is located at an amphiphilic interface, its conformation changes, the "lid" opens, and the active center is exposed. Therefore, lipase activity is regulated by the hydrophilic / hydrophobic microenvironment of the support. A suitable hydrophilic / hydrophobic microenvironment can improve the interaction between the support and lipase, enhancing enzyme activity and stability.

[0003] In order to regulate the hydrophilic / hydrophobic microenvironment of the carrier and improve the activity and stability of immobilized lipase, researchers have developed a variety of hydrophobic modification methods for immobilized lipase carriers, mainly the following:

[0004] CN 106701729 A discloses an immobilized enzyme using a polypeptide-modified amino resin as a carrier and its preparation method. Hydrophobic amino acids are chemically grafted onto the amino resin to form hydrophobic polypeptide fragments of varying chain lengths, creating a stable hydrophobic environment on the carrier surface. This results in a functional carrier with a relatively hydrophobic surface environment, upon which lipase can be immobilized by physical adsorption. The amino acid-modified carriers immobilized enzymes, amino resin-leucine residue (length 10)-lipase and amino resin-phenylalanine residue (length 10)-lipase, showed residual enzyme activities of 92.75% and 80.86%, respectively, after 12 days of storage. After 21 days, the residual enzyme activities remained at 36.66% and 50.66% of the initial values, respectively, demonstrating excellent stability. However, this method introduces toxic chemical condensation reagents during carrier modification, making it difficult to apply in the food industry.

[0005] CN 116463327 A discloses an immobilized lipase using an acyl chloride-modified amino resin as a carrier, as well as its preparation and application. The amino resin is hydrophobically modified with alkyl acyl chlorides of varying carbon chain lengths, the lipase is physically adsorbed onto the resulting acyl chloride-modified amino resin, and then cross-linked using the natural cross-linking agent genipin. The resulting immobilized lipase has an enzyme activity recovery rate of 176%, nearly nine times that of lipase immobilized by adsorption on unmodified resin. However, this method introduces toxic alkyl acyl chlorides during carrier modification, making it difficult to apply in the food industry.

[0006] CN 119391688 A discloses a highly stable immobilized Antarctic Candida lipase B, its preparation method, and application. ECR1030 macroporous adsorption resin is hydrophobically modified with a silane coupling agent to produce a silanized resin. The silanized resin is then functionalized with dialdehyde cellulose to form an alkyl-aldehyde-modified resin for immobilizing the Antarctic Candida lipase B. After interfacial activation on the surface of the alkyl-aldehyde-modified resin, the Antarctic Candida lipase B covalently bonds to the aldehyde groups on the surface of the alkyl-aldehyde-modified resin. The resulting immobilized Antarctic Candida lipase B can be reused 50 times with virtually no loss of activity. However, this method requires high temperatures (80°C-95°C) during the carrier modification process, consumes significant energy, and is complex.

[0007] CN 118755788 A discloses a method for catalyzing the synthesis of phytosterol ester compounds using a bifunctional modified macroporous resin immobilized lipase. Stearic acid is hydrophobically modified on an amino resin using a chemical coupling agent. Glutaraldehyde is used to activate the remaining amino groups on the surface. The immobilized enzyme is then immobilized using a Schiff base reaction with the Candida plicata lipase and a hydrophobic interaction between the C18 alkyl group and the enzyme. The immobilized enzyme is applied to the esterification of phytosterols with fatty acids, achieving a phytosterol ester yield of 95.7% in 48 hours. The immobilized enzyme exhibits high catalytic activity and good reusability. After six consecutive reaction cycles, the enzyme still maintains 76.2% of its initial activity. However, this method introduces highly toxic chemical coupling agents and glutaraldehyde during the carrier modification process, making it difficult to apply to the food industry. Summary of the Invention

[0008] The purpose of the present invention is to provide a green, simple and low-cost method for hydrophobic modification of amino resin, which can accurately regulate the hydrophilic / hydrophobic microenvironment of the carrier to improve the activity and stability of immobilized lipase.

[0009] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0010] In one aspect, the present invention provides a method for preparing immobilized lipase using a grafted modified amino resin as a carrier, comprising the following steps:

[0011] Step S1: dissolving tannic acid in alkaline buffer A, adding amino resin, performing oxidation reaction at room temperature, and filtering and drying to obtain a first modified amino resin;

[0012] Step S2: adding the fatty amine and the first modified amino resin to an ethanol solution respectively to carry out a grafting reaction, and filtering and drying to obtain a second modified amino resin;

[0013] Step S3: adding the second modified amino resin to the lipase solution, filtering out the liquid after adsorption, rinsing with buffer solution B, and obtaining the immobilized lipase after drying.

[0014] In the above technical solution, the concentration of tannic acid dissolved in buffer A in step S1 is 1 mg / mL-5 mg / mL. Preferably, the concentration of tannic acid dissolved in buffer A is 2 mg / mL.

[0015] In the above technical solution, in step S1, buffer A is sodium bicarbonate buffer or Tris-hydrochloric acid buffer or HEPES buffer, preferably, buffer A is sodium bicarbonate buffer; the pH of buffer A is 8-9, preferably, the pH of buffer A is 8.5.

[0016] In the above technical solution, the oxidation reaction time in step S1 is 6-24 h, preferably, the oxidation reaction time is 12 h.

[0017] In the above technical solution, the model of the amino resin in step S1 is one of LX-1000HAA, LX-1000EPN, LX-1000EPHA, LX-1000NH and LX-1000EA.

[0018] In the above technical solution, the fatty amine in step S2 includes one or more of hexylamine, octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, and octadecylamine. Preferably, the fatty amine is octadecylamine.

[0019] In the above technical solution, the amount of fatty amine added in step S2 is 0.2 mmol-1 mmol; preferably, the amount of fatty amine added is 0.4 mmol; the concentration of the ethanol solution is 50%-100%; preferably, the concentration of the ethanol solution is 70%; the grafting reaction temperature is 40°C-60°C, preferably, the grafting reaction temperature is 50°C; the grafting reaction time is 4-8 h, preferably, the grafting reaction time is 6 h.

[0020] In the above technical solution, the lipase solution in step S3 is prepared by dissolving the lipase in buffer solution B. Preferably, buffer solution B is a phosphate solution.

[0021] In the above technical solution, the mass volume ratio of the second modified amino resin to the lipase solution in step S3 is 1 g: 5-25 mL. Preferably, the mass volume ratio of the second modified amino resin to the lipase solution is 1 g: 10 mL.

[0022] In the above technical solution, the pH of the lipase solution in step S3 is 5-10, preferably 7; the adsorption time is 2-10 h, preferably 8 h; the adsorption temperature is 25-45 °C, preferably 35 °C.

[0023] In the above technical solution, the lipase is Rhizomucor miehei lipase.

[0024] Another aspect of the present invention provides an immobilized lipase prepared using a grafted modified amino resin as a carrier, which is prepared according to any of the above methods.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The activity and stability of the immobilized lipase are improved by precisely designing the hydrophilic / hydrophobic microenvironment of the immobilized lipase carrier. First, a layer of oxidized tannic acid coating is formed on the amino resin by utilizing the oxidative self-polymerization property of tannic acid in an alkaline environment. The Schiff base and Michael addition reactions between the quinone active groups on the oxidized tannic acid coating and the amino groups of the fatty amine are used to anchor fatty amines of different numbers and carbon chain lengths on the surface of the tannic acid coating for hydrophobic modification to obtain a second modified amino resin. Subsequently, the lipase is immobilized on the second modified amino resin by physical adsorption. The lipase undergoes interfacial activation at the amphiphilic interface on the second modified amino resin, and a portion of the lipase is covalently bound to the quinone group in the activated state, thereby obtaining an immobilized lipase with high activity and stability. Only ethanol solution is used in the modification process of the amino resin of the present invention, which is a safe and environmentally friendly method for preparing immobilized lipase. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1This is a technical roadmap for the method of preparing immobilized lipase using grafted modified amino resin as a carrier of the present invention.

[0029] Figure 2 This is a schematic diagram of the principle of grafting modified amino resins of the present invention.

[0030] Figure 3 The effect of enzyme addition amount on enzyme activity and immobilization efficiency.

[0031] Figure 4 The effect of enzyme solution pH on enzyme activity and immobilization efficiency.

[0032] Figure 5 The effect of adsorption time on enzyme activity and immobilization efficiency.

[0033] Figure 6 The effect of adsorption temperature on enzyme activity and immobilization efficiency.

[0034] Figure 7 To characterize the thermal stability of immobilized lipase.

[0035] Figure 8 To characterize the storage stability of immobilized lipase. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0037] like Figure 1 As shown, the present invention proposes a method for preparing immobilized lipase using a grafted modified amino resin as a carrier, comprising the following steps:

[0038] Step S1: dissolving tannic acid in alkaline buffer A, adding amino resin, stirring at room temperature to carry out oxidation reaction, and filtering and drying to obtain a first modified amino resin;

[0039] Step S2: adding the fatty amine and the first modified amino resin to an ethanol solution respectively to carry out a grafting reaction, and filtering and drying to obtain a second modified amino resin;

[0040] Step S3: adding the second modified amino resin to the lipase solution, filtering out the liquid after adsorption, rinsing with buffer solution B, and obtaining the immobilized lipase after drying.

[0041] Combine Figure 2The principle of the present method for preparing immobilized lipase is as follows: lipase, a typical interfacially activated enzyme, requires a suitable hydrophilic / hydrophobic microenvironment for interfacial activation. However, amino resins, due to their abundant surface amino groups, exhibit hydrophilicity. Direct use as immobilized enzyme carriers fails to effectively activate lipase, resulting in low enzyme activity. Therefore, the amino resins need to be hydrophobically modified to create a suitable hydrophilic / hydrophobic microenvironment and improve enzyme activity and stability. In step S1, tannic acid self-polymerizes under alkaline conditions to form an oxidized tannic acid coating. Simultaneously, since its phenolic hydroxyl groups are easily oxidized to form quinones in an alkaline environment, the quinone groups, acting as active groups, can undergo Schiff base and Michael addition reactions with amino groups on the amino resin to form covalent bonds, thereby helping the tannic acid to quickly and tightly form a layer of oxidized tannic acid coating on the amino resin. In step S2, hydrophobic fatty amines of varying numbers and carbon chain lengths are anchored to the surface of the tannic acid coating using Schiff base and Michael addition reactions between the remaining quinone active groups on the oxidized tannic acid coating and the amino groups of the fatty amines, thereby forming immobilized enzyme carriers with varying hydrophilic / hydrophobic properties. In step S3, the lipase is immobilized on the second modified amino resin by physical adsorption. The lipase undergoes interfacial activation at the amphiphilic interface on the second modified amino resin, and a portion of the lipase is covalently bound to the quinone group in the activated state, thereby obtaining an immobilized lipase with high activity and stability. The present invention innovatively introduces an oxidized tannic acid coating as a multifunctional platform, and performs hydrophobic modification and enzyme immobilization on it through Schiff base and Michael addition reactions without the need for introducing additional cross-linking agents. By controlling the chain length and grafting density of the fatty amine, an amphiphilic interface with adjustable hydrophilicity / hydrophobicity is constructed on the amino resin, providing a suitable immobilization microenvironment for the lipase, thereby improving the activity and stability of the immobilized lipase.

[0042] The enzyme activity detection method in the following examples is as follows:

[0043] Definition of enzyme activity: Under certain reaction conditions, the amount of enzyme required for the immobilized enzyme to catalyze the esterification of lauric acid and n-propanol to produce 1 μmol of propyl laurate per minute is defined as one enzyme activity unit (U).

[0044] Determination method: 80.1 g of lauric acid, 24 g of n-propanol, and 3.2 g of distilled water were premixed. 5.36 g of the premixed substrate was placed in a 50 mL ground-mouth Erlenmeyer flask with a stopper and shaken in a constant-temperature water bath at 200 rpm and 60°C for 10 minutes. Approximately 15 mg of immobilized lipase was then added. After a 20-minute reaction, 30 μL of the reaction product was immediately dissolved in 970 μL of n-heptane and the lauric acid and propyl laurate contents were determined by gas chromatography (GC). Enzyme activity was calculated using the following formula:

[0045]

[0046] Where N is the amount of lauric acid in the substrate (mol); C is the ratio of lauric acid converted to propyl laurate; W is the mass of the enzyme (g); and t is the reaction time (min).

[0047] The fixed efficiency is calculated as follows:

[0048]

[0049] Where C0 is the protein content of the enzyme solution before immobilization, and C1 is the protein content of the enzyme solution after immobilization.

[0050] The main raw materials and reagents in the examples are as follows: tannic acid: purity 98%, Maclean's reagent; octadecylamine: purity 99%, Maclean's reagent; hexadecylamine: purity 95%, Maclean's reagent; tetradecylamine: purity 96%, Maclean's reagent; dodecylamine: purity 98%, Maclean's reagent; decylamine: purity 98%, Maclean's reagent; octylamine: purity 99%, Maclean's reagent; hexylamine: purity 99%, Maclean's reagent; Rhizomucor miehei lipase: enzyme activity 1007 U / mL, Qingdao Weilan Biotechnology Co., Ltd.

[0051] Buffer A was prepared by weighing 50 mmol of one of sodium bicarbonate, Tris (tris(hydroxymethylaminomethane), and HEPES (hydroxyethylpiperazineethanesulfonic acid), dissolving it in 800 mL of distilled water, adjusting the pH to the corresponding value with sodium hydroxide solution, and then diluting the volume to 1 L.

[0052] Buffer B was prepared as follows: 50 mmol of potassium dihydrogen phosphate and dipotassium hydrogen phosphate were weighed and dissolved in 800 mL of distilled water. The pH value was adjusted to the corresponding value with sodium hydroxide solution, and the volume was made up to 1 L. Example 1

[0053] 10 g of wet LX-1000HAA amino resin was weighed and dissolved in 200 mL of sodium bicarbonate buffer (pH 8.5, 50 mM). 0.4 g of tannic acid (the concentration of tannic acid in buffer A was 2 mg / mL) was added and stirred at room temperature for 12 h. The mixture was filtered and dried under vacuum to obtain the first modified amino resin. 1 g of the first modified amino resin was added to 25 mL of 70% ethanol (v / v). 0.4 mmol of octadecylamine, hexadecylamine, tetradecylamine, dodecylamine, decylamine, octylamine, and hexylamine were added, respectively. The reaction was continued at 50°C for 6 h. The reaction was filtered and dried under vacuum to obtain the second modified amino resin. 2.5 g of Rhizomucor miehei lipase was dissolved in 50 mL of phosphate buffer (pH 7, 50 mM). After centrifugation, the supernatant was collected to obtain the lipase solution. Take 4 mL of the above lipase solution, add 0.2 g of the second modified amino resin, and immobilize at 30°C for 10 h. Filter and separate the immobilized lipase. Wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate. Dry the washed immobilized product in a vacuum at 40°C for 4 h.

[0054] The effects of various fatty amine types on immobilization efficiency and enzyme activity were compared and analyzed to determine the optimal fatty amine type. As shown in Table 1, enzyme activity increased with increasing carbon chain length of the fatty amine. Therefore, using octadecylamine as a hydrophobic modifier was most effective in enhancing enzyme activity, reaching a maximum enzyme activity of 10,823.54 U / g and an immobilization efficiency of 52.35%.

[0055] Example 2

[0056] 10 g of wet LX-1000HAA amino resin was weighed and dissolved in 200 mL of sodium bicarbonate buffer (pH 8.5, 50 mM). 0.4 g of tannic acid (the concentration of tannic acid in buffer A was 2 mg / mL) was added and stirred at room temperature for 12 h. The mixture was filtered and dried under vacuum to obtain the first modified amino resin. 1 g of the first modified amino resin was added to 25 mL of 70% ethanol solution (v / v). 0.2 mmol, 0.4 mmol, 0.6 mmol, 0.8 mmol, and 1.0 mmol of octadecylamine were added, respectively. The reaction was continued at 50°C for 6 h. The mixture was filtered and dried under vacuum to obtain the second modified amino resin. 2.5 g of Rhizomucor miehei lipase was dissolved in 50 mL of phosphate buffer (pH 7, 50 mM). After centrifugation, the supernatant was collected to obtain the lipase solution. Take 4 mL of the above lipase solution, add 0.2 g of the second modified amino resin, and immobilize at 30°C for 10 h. Filter and separate the immobilized lipase. Wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate. Dry the washed immobilized product in a vacuum at 40°C for 4 h.

[0057] The effects of the addition amount of octadecylamine on immobilization efficiency and enzyme activity were compared and analyzed to determine the optimal addition amount. As shown in Table 2, with increasing addition amount of octadecylamine, the enzyme activity showed a trend of first increasing and then decreasing. When the addition amount of octadecylamine was 0.4 mmol, the highest immobilization efficiency and enzyme activity were achieved, reaching 51.51% and 10781.58 U / g, respectively.

[0058] Example 3

[0059] 10 g of wet LX-1000HAA amino resin was weighed and dissolved in 200 mL of sodium bicarbonate buffer (pH 8.5, 50 mM). 0.4 g of tannic acid (the concentration of tannic acid in buffer A was 2 mg / mL) was added and stirred at room temperature for 12 h. The mixture was filtered and dried under vacuum to obtain the first modified amino resin. 1 g of the first modified amino resin was added to 25 mL of 70% ethanol (v / v) and 0.4 mmol of octadecylamine. The reaction was continued at 50°C for 6 h, filtered, and dried under vacuum to obtain the second modified amino resin. 2.5 g of Rhizomucor miehei lipase was dissolved in 50 mL of phosphate buffer (pH 7, 50 mM). After centrifugation, the supernatant was collected to obtain the lipase solution. 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of the above lipase solution were taken, respectively, and 0.2 g of the second modified amino resin was added. The mixture was immobilized at 30°C for 8 h. The immobilized lipase was separated by filtration and washed with phosphate buffer until no protein remained in the filtrate. The immobilized product after washing was vacuum dried at 40°C for 4 h.

[0060] Compare and analyze the effects of enzyme addition on immobilization efficiency and enzyme activity to determine the optimal enzyme addition amount. Figure 3 As shown in the figure, with the increase of enzyme addition amount, the enzyme activity showed a trend of first increasing and then decreasing. When the enzyme addition amount was 10 mL / g, the enzyme activity was the highest, which could reach 10909.62 U / g, and the immobilization efficiency was 69.83%. Example 4

[0061] 10 g of wet LX-1000HAA amino resin was weighed and dissolved in 200 mL of sodium bicarbonate buffer (pH 8.5, 50 mM). 0.4 g of tannic acid (the concentration of tannic acid in buffer A was 2 mg / mL) was added and stirred at room temperature for 12 h. The mixture was filtered and dried under vacuum to obtain the first modified amino resin. 1 g of the first modified amino resin was added to 25 mL of 70% ethanol (v / v) and 0.4 mmol of octadecylamine. The reaction was continued at 50°C for 6 h, filtered, and dried under vacuum to obtain the second modified amino resin. 2.5 g of Rhizomucor miehei lipase was dissolved in 50 mL of 50 mM phosphate buffer at pH 5, 6, 7, 8, 9, or 10, respectively. The supernatant was collected after centrifugation to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, and immobilize at 30°C for 10 h. Filter and separate the immobilized lipase. Wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate. Dry the washed immobilized product in a vacuum at 40°C for 4 h.

[0062] Compare and analyze the effect of enzyme solution pH on immobilization efficiency and enzyme activity to determine the optimal enzyme addition amount. Figure 4 As shown in the figure, with the increase of pH of the enzyme solution, the enzyme activity showed a trend of first increasing and then decreasing. When the pH of the enzyme solution was 7, the enzyme activity was the highest, which could reach 11002.84 U / g, and the immobilization efficiency was 68.12%. Example 5

[0063] 10 g of wet LX-1000HAA amino resin was weighed and dissolved in 200 mL of sodium bicarbonate buffer (pH 8.5, 50 mM). 0.4 g of tannic acid (the concentration of tannic acid in buffer A was 2 mg / mL) was added and stirred at room temperature for 12 h. The mixture was filtered and dried under vacuum to obtain the first modified amino resin. 1 g of the first modified amino resin was added to 25 mL of 70% ethanol (v / v) and 0.4 mmol of octadecylamine. The reaction was continued at 50°C for 6 h, filtered, and dried under vacuum to obtain the second modified amino resin. 2.5 g of Rhizomucor miehei lipase was dissolved in 50 mL of phosphate buffer (pH 7, 50 mM). After centrifugation, the supernatant was collected to obtain the lipase solution. To 2 mL of the above lipase solution, 0.2 g of the second modified amino resin was added and immobilized at 30°C for 2, 4, 6, 8, and 10 h, respectively. The immobilized lipase was separated by filtration and washed with phosphate buffer until no protein remained in the filtrate. The washed immobilized product was vacuum dried at 40°C for 4 h.

[0064] Compare and analyze the effect of enzyme solution immobilization time on immobilization efficiency and enzyme activity to determine the optimal immobilization time. Figure 5 As shown in the figure, with the increase of immobilization time, the enzyme activity and immobilization efficiency showed an increasing trend, and then gradually reached a balance. When the immobilization time of the enzyme solution was 8 h, the enzyme activity reached the highest, and the enzyme activity could reach 11363.91 U / g. At this time, the immobilization efficiency was 64.32%. Example 6

[0065] 10 g of wet LX-1000HAA amino resin was weighed and dissolved in 200 mL of sodium bicarbonate buffer (pH 8.5, 50 mM). 0.4 g of tannic acid (the concentration of tannic acid in buffer A was 2 mg / mL) was added and stirred at room temperature for 12 h. The mixture was filtered and dried under vacuum to obtain the first modified amino resin. 1 g of the first modified amino resin was added to 25 mL of 70% ethanol (v / v) and 0.4 mmol of octadecylamine. The reaction was continued at 50°C for 6 h, filtered, and dried under vacuum to obtain the second modified amino resin. 2.5 g of Rhizomucor miehei lipase was dissolved in 50 mL of phosphate buffer (pH 7, 50 mM). After centrifugation, the supernatant was collected to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, and immobilize at 25°C, 30°C, 35°C, 40°C, and 45°C for 8 h, respectively. The immobilized lipase is separated by filtration and washed with phosphate buffer until no protein remains in the filtrate. The washed immobilized product is vacuum dried at 40°C for 4 h.

[0066] Compare and analyze the effect of enzyme solution immobilization temperature on immobilization efficiency and enzyme activity to determine the optimal immobilization temperature. Figure 6 As shown in the figure, with the increase of immobilization temperature, the enzyme activity and immobilization efficiency showed a trend of first increasing and then decreasing. When the immobilization temperature of the enzyme solution was 35 ℃, the enzyme activity was the highest, reaching 11638.53 U / g, and the immobilization efficiency was 68.58%. Example 7

[0067] 10 g of wet LX-1000EPN amino resin was weighed and dissolved in 200 mL of sodium bicarbonate buffer (pH 8.5, 50 mM). 0.2 g of tannic acid (1 mg / mL tannic acid dissolved in buffer A) was added and stirred at room temperature for 24 h. The mixture was filtered and dried under vacuum to obtain the first modified amino resin. 1 g of the first modified amino resin was added to 25 mL of 70% ethanol (v / v) and 0.4 mmol of octadecylamine. The reaction was continued at 50°C for 6 h, filtered, and dried under vacuum to obtain the second modified amino resin. 2.5 g of Rhizomucor miehei lipase was dissolved in 50 mL of phosphate buffer (pH 7, 50 mM). After centrifugation, the supernatant was collected to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, and fix them at 35°C for 8 h. Filter and separate the immobilized lipase. Wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate. Dry the washed immobilized product in a vacuum at 40°C for 4 h.

[0068] The properties of the immobilized lipase were characterized, and the results showed that the immobilization efficiency was 65.52% and the enzyme activity was 10325.32 U / g. Example 8

[0069] 10 g of wet LX-1000HAA amino resin was weighed and dissolved in 200 mL of Tris-HCl buffer (pH 8, 50 mM). 0.4 g of tannic acid (the concentration of tannic acid in buffer A was 2 mg / mL) was added and stirred at room temperature for 18 h. The mixture was filtered and dried under vacuum to obtain the first modified amino resin. 1 g of the first modified amino resin was added to 25 mL of 90% ethanol (v / v) and 0.4 mmol of octadecylamine. The reaction was continued at 40°C for 8 h, filtered, and dried under vacuum to obtain the second modified amino resin. 2.5 g of Rhizomucor miehei lipase was dissolved in 50 mL of phosphate buffer (pH 7, 50 mM). After centrifugation, the supernatant was collected to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, and fix them at 35°C for 8 h. Filter and separate the immobilized lipase. Wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate. Dry the washed immobilized product in a vacuum at 40°C for 4 h.

[0070] The properties of the immobilized lipase were characterized, and the results showed that the immobilization efficiency was 62.43% and the enzyme activity was 9823.73 U / g. Example 9

[0071] 10 g of wet LX-1000NH amino resin was weighed and dissolved in 200 mL of HEPES buffer (pH 9, 50 mM). 0.6 g of tannic acid (3 mg / mL tannic acid dissolved in buffer A) was added and stirred at room temperature for 10 h. The mixture was filtered and dried under vacuum to obtain the first modified amino resin. 1 g of the first modified amino resin was added to 25 mL of 50% ethanol (v / v) and 0.4 mmol of octadecylamine. The reaction was continued at 60°C for 4 h, filtered, and dried under vacuum to obtain the second modified amino resin. 2.5 g of Rhizomucor miehei lipase was dissolved in 50 mL of phosphate buffer (pH 7, 50 mM). After centrifugation, the supernatant was collected to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, and fix them at 35°C for 8 h. Filter and separate the immobilized lipase. Wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate. Dry the washed immobilized product in a vacuum at 40°C for 4 h.

[0072] The properties of the immobilized lipase were characterized, and the results showed that the immobilization efficiency was 64.23% and the enzyme activity was 9547.68 U / g. Example 10

[0073] 10 g of wet LX-1000EA amino resin was weighed and dissolved in 200 mL of HEPES buffer (pH 8.5, 50 mM). 0.8 g of tannic acid (4 mg / mL tannic acid dissolved in buffer A) was added and stirred at room temperature for 8 h. The mixture was filtered and dried under vacuum to obtain the first modified amino resin. 1 g of the first modified amino resin was added to 25 mL of 80% ethanol (v / v) and 0.4 mmol of octadecylamine. The reaction was continued at 45°C for 7 h. The reaction was filtered and dried under vacuum to obtain the second modified amino resin. 2.5 g of Rhizomucor miehei lipase was dissolved in 50 mL of phosphate buffer (pH 7, 50 mM). After centrifugation, the supernatant was collected to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, and fix them at 35°C for 8 h. Filter and separate the immobilized lipase. Wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate. Dry the washed immobilized product in a vacuum at 40°C for 4 h.

[0074] The properties of the immobilized lipase were characterized, and the results showed that the immobilization efficiency was 64.57% and the enzyme activity was 10435.97 U / g. Example 11

[0075] 10 g of wet LX-1000 EPHA amino resin was weighed and dissolved in 200 mL of Tris-HCl buffer (pH 8.5, 50 mM). 1 g of tannic acid (5 mg / mL tannic acid dissolved in buffer A) was added and stirred at room temperature for 6 h. The mixture was filtered and dried under vacuum to obtain the first modified amino resin. 1 g of the first modified amino resin was added to 25 mL of 100% ethanol (v / v) and 0.4 mmol of octadecylamine. The reaction was continued at 55°C for 5 h, filtered, and dried under vacuum to obtain the second modified amino resin. 2.5 g of Rhizomucor miehei lipase was dissolved in 50 mL of phosphate buffer (pH 7, 50 mM). After centrifugation, the supernatant was collected to obtain the lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the second modified amino resin, and fix them at 35°C for 8 h. Filter and separate the immobilized lipase. Wash the immobilized lipase with phosphate buffer until there is no protein residue in the filtrate. Dry the washed immobilized product in a vacuum at 40°C for 4 h.

[0076] The properties of the immobilized lipase were characterized, and the results showed that the immobilization efficiency was 65.52% and the enzyme activity was 9769.58 U / g. Comparative Example 1

[0077] 4 mL of the lipase solution in Example 1 was added with 0.2 g of vacuum-dried LX-1000HAA amino resin and immobilized at 30°C for 10 h. The immobilized lipase was separated by filtration and washed with phosphate buffer until no protein remained in the filtrate. The washed immobilized product was vacuum-dried at 40°C for 4 h.

[0078] The properties of the immobilized lipase were characterized, and the results showed that the immobilization efficiency was 31.91% and the enzyme activity was 5995.66 U / g. Comparative Example 2

[0079] Weigh 10 g of wet LX-1000HAA resin in 200 mL of sodium bicarbonate buffer (pH 8.5, 50 mM), add 0.4 g of tannic acid (the concentration of tannic acid dissolved in buffer A is 2 mg / mL), stir at room temperature for 12 h, filter, and vacuum dry to obtain the first modified amino resin. Add 0.2 g of the first modified amino resin to 4 mL of the lipase solution from Example 1 and immobilize at 30°C for 10 h. Filter and isolate the immobilized lipase. Wash the immobilized lipase with phosphate buffer until no protein remains in the filtrate. Dry the washed immobilized product in vacuum at 40°C for 4 h.

[0080] The properties of the immobilized lipase were characterized, and the results showed that the immobilization efficiency was 35.14% and the enzyme activity was 6895.43 U / g.

[0081] Table 3 shows a comparison of the characterization results of Examples 1-11 and Comparative Examples 1 and 2. Compared to amino resin, the modified amino resin significantly improved the enzymatic activity and immobilization efficiency of immobilized lipase. Under optimal experimental conditions, the enzyme activity increased by up to 94.12% compared to amino resin.

[0082]

[0083] The second modified amino resin immobilized lipase grafted with octadecylamine in Example 1 and the amino resin immobilized lipase in Comparative Example 1 were incubated at 40°C and 60°C for 10 h, respectively, and samples were taken regularly to measure the enzyme activity. Figure 7As shown in the results, the activity of the amino resin-immobilized lipase decreased more significantly at both 40°C and 60°C than that of the second modified amino resin-immobilized lipase. After incubation at 60°C for 10 h, the second modified amino resin-immobilized lipase still retained 79.15% of its initial activity, while the amino resin-immobilized lipase retained only 57.92%. These results indicate that the second modified amino resin-immobilized lipase exhibits significantly improved thermal stability compared to the amino resin-immobilized lipase.

[0084] The second modified amino resin immobilized lipase grafted with octadecylamine in Example 1 and the amino resin immobilized lipase in Comparative Example 1 were stored at 4°C and samples were taken regularly to measure the enzyme activity. Figure 8 As shown in the results, at 4°C, the activity of the amino resin-immobilized lipase decreased significantly more than that of the second modified amino resin-immobilized lipase. After 30 days of storage at 4°C, the second modified amino resin-immobilized lipase still retained 95.35% of its initial activity, while the amino resin-immobilized lipase only retained 80.13%. These results indicate that the storage stability of the second modified amino resin-immobilized lipase is significantly improved compared to that of the amino resin-immobilized lipase.

[0085] The method for detecting protein residues in the filtrate in each embodiment of the present invention and the comparative example adopts the Coomassie Brilliant Blue method; other treatment methods not described in detail, such as filtration, vacuum drying, etc., can be treated according to conventional treatment methods in the industry and will not be described in detail in this application.

[0086] 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 changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for preparing immobilized lipase using a grafted modified amino resin as a carrier, characterized in that: The following steps are involved: Step S1: dissolving tannic acid in an alkaline buffer solution, adding an amino resin, performing an oxidation reaction at room temperature, and filtering and drying to obtain a first modified amino resin; Step S2: adding the fatty amine and the first modified amino resin to an ethanol solution respectively to carry out a grafting reaction, and filtering and drying to obtain a second modified amino resin; Step S3: adding the second modified amino resin to the lipase solution, filtering out the liquid after adsorption, eluting with a buffer solution, and obtaining the immobilized lipase after drying.

2. The method for preparing immobilized lipase using a grafted modified amino resin as a carrier according to claim 1, characterized in that: In step S1, the concentration of tannic acid dissolved in alkaline buffer is 1 mg / mL-5 mg / mL.

3. The method for preparing immobilized lipase using a grafted modified amino resin as a carrier according to claim 2, characterized in that: The alkaline buffer in step S1 is sodium bicarbonate buffer, Tris-hydrochloric acid buffer or HEPES buffer, and the pH of the alkaline buffer is 8-9.

4. The method for preparing immobilized lipase using a grafted modified amino resin as a carrier according to claim 1, characterized in that: The model of the amino resin in step S1 is one of LX-1000HAA, LX-1000EPN, LX-1000EPHA, LX-1000NH and LX-1000EA.

5. The method for preparing immobilized lipase using a grafted modified amino resin as a carrier according to claim 1, characterized in that: The fatty amine in step S2 includes one or more of hexylamine, octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, and octadecylamine.

6. The method for preparing immobilized lipase using a grafted modified amino resin as a carrier according to claim 5, characterized in that: In step S2, the amount of fatty amine added is 0.2 mmol-1 mmol; the concentration of the ethanol solution is 50%-100%; the grafting reaction temperature is 40°C-60°C; and the grafting reaction time is 4-8 hours.

7. The method for preparing immobilized lipase using a grafted modified amino resin as a carrier according to claim 1, characterized in that: In step S3, the mass volume ratio of the second modified amino resin to the lipase solution is 1 g: 5-25 mL.

8. The method for preparing immobilized lipase using a grafted modified amino resin as a carrier according to claim 6, characterized in that: In step S3, the pH of the lipase solution is 5-10, the adsorption time is 2-10 h, and the adsorption temperature is 25-45°C.

9. The method for preparing immobilized lipase using a grafted modified amino resin as a carrier according to claim 1, characterized in that: The lipase is Rhizomucor miehei lipase.

10. An immobilized lipase prepared using a grafted modified amino resin as a carrier, characterized in that: It is prepared according to the method according to any one of claims 1 to 9.

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