Preparation method and application of copper-based metal organic material in-situ synthesis immobilized lipase

Abstract

The invention discloses a preparation method of a copper-based metal organic material, which is used for in-situ synthesis of immobilized lipase. The preparation method comprises the following steps: firstly, preparing an acetate buffer solution with a certain concentration and a certain pH value as a solvent; secondly, 2-aminoterephthalic acid (H2ATA) is taken and added into the acetate buffer solution, and lipase is added after 2-aminoterephthalic acid (H2ATA) is fully dissolved; thirdly, dissolving a certain mass of copper salt into the acetate buffer solution, and performing ultrasonic treatment to obtain a copper ion solution; and finally, dropwise adding the copper ion solution into the H2ATA-lipase solution while stirring, and carrying out suction filtration and drying to obtain the copper-based metal organic material immobilized lipase. Compared with free enzyme, the immobilized lipase has the advantages that the enzyme activity is improved by 10% or above, the loading capacity is 400 mg / g or above, 63.5% of the original activity can still be reserved after the immobilized lipase is repeatedly used for 6 times or above, and the preparation method is simple, convenient and suitable for industrial use.

Description

Technical Field

[0001] The invention belongs to the technical field of immobilized enzymes and relates to a preparation method and application of immobilized lipase synthesized in situ by using copper-based metal organic materials. Background Art

[0002] Lipase is a special type of esterase that can hydrolyze triglycerides into fatty acids, diglycerides and glycerol, etc. Its natural substrate is generally a long-chain fatty acyl ester that is insoluble in water. As an important biocatalyst, lipase can catalyze the hydrolysis and synthesis reactions of different substrates. This catalytic property makes it applicable in the fields of food processing, dairy products, feed industry, bioenergy, biomedicine, detergents, textiles, etc., and its application in food is particularly extensive. However, free lipase has strict requirements on reaction conditions, is sensitive to ambient temperature and has poor recovery rate. This results in low reuse rate, increases the cost of lipase, and thus limits its industrial application.

[0003] Immobilizing the enzyme on a carrier can increase the enzyme's stability to acid, alkali and temperature and its reusability. Common immobilization methods include physical adsorption, embedding, covalent bonding and cross-linking. Physical adsorption and embedding are inefficient, covalent bonding is only applicable to smaller substrates, and cross-linking can lead to inactivation of enzyme proteins. Metal organic materials are compounds with different dimensions formed by coordination reactions between metal ions and organic ligands. They have the advantages of high specific surface area, good mechanical strength, uniform and adjustable pore size, and can be modified with active groups. Due to their excellent structural characteristics, metal organic materials have gradually shown excellent application prospects in the field of immobilized enzymes. Therefore, we prepared a copper-based metal organic material immobilized lipase.

[0004] In recent years, many methods for preparing metal organic material immobilized enzymes have been developed at home and abroad. For example, patent CN110129290A discloses a method for preparing metal organic framework material immobilized enzymes by first covalently binding laccase with metal copper ions and then combining with trimesic acid. Patent CN114540334A first synthesizes metal skeleton organic materials using zirconium tetrachloride and terephthalic acid in an organic phase, and then mixes with lipase in PBS buffer to prepare immobilized lipase. Patent CN115044577A discloses a method for preparing immobilized lipase by synthesizing ZIF-8 in situ encapsulation of lipase using zinc acetate and 2-methylimidazole. Patent CN114958818A adopts a one-pot synthesis method to mix aspartate aminotransferase, branched-chain amino acid aminotransferase, metal source and organic ligand solution at room temperature to prepare metal organic framework material immobilized enzymes. Patent CN114736895A first reacts a weak ligand, a metal salt and a biological enzyme in a solution system, and then immerses the resulting MOF composite material in a neutral solution system containing a strong ligand to react, thereby obtaining an immobilized enzyme.

[0005] The metal organic materials prepared by existing research have shown excellent performance in the field of immobilized enzymes, but in the process of preparing metal organic materials, metal ions are used to enhance enzyme activity, so that immobilized enzymes with better activity can be further prepared. When the metal organic material is formed in the present invention, copper ions spontaneously bind to lipase, open the α-helix outside the active center of the lipase, expose the internal structure of the lipase, and form an electrophilic domain around the serine residue in the active center, thereby causing conformational changes in the lipase, thereby enhancing enzyme activity. The metal organic material wraps the lipase in it while nucleating and growing, and obtains a metal organic material immobilized lipase. This immobilized lipase has a high loading rate, reuse rate and stability, and has a wide range of application possibilities in the fields of feed industry, bioenergy, biomedicine, detergents, textiles, etc. Summary of the invention

[0006] Preparation method and application of in-situ synthesis of immobilized lipase using copper-based metal organic materials The reagents used in the present invention are from a reagent company. The technical solution adopted by the present invention is: A copper-based metal organic material is prepared by adding a copper salt to a buffer solution of a certain pH and a certain concentration to prepare a copper ion solution of a certain concentration, and ultrasonically treating the solution until the solution is homogeneous; 2-aminoterephthalic acid is added to the buffer solution to prepare an organic ligand solution of a certain concentration, and after stirring and ultrasonicating, a certain amount of lipase is dissolved to form an organic ligand-lipase solution, and the copper ion solution is added dropwise while stirring. The solution is filtered, the solid is taken, and the solid is washed with water and dried. The enzyme-immobilized copper-based metal organic material is obtained.

[0007] Furthermore, the buffer is preferably at least one of acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, and citric acid-sodium hydroxide buffer, and the pH of the buffer is 7-8.

[0008] Furthermore, the copper salt is preferably at least one of copper sulfate, copper acetate and copper chloride.

[0009] Furthermore, the Cu 2+ The ratio with H2ATA is 0.42 / 1~1.17 / 1 g / g.

[0010] Furthermore, the Cu 2+ The ratio to enzyme protein is 0.55 / 1~2.55 / 1 g / g.

[0011] Furthermore, after the organic ligand-lipase solution is prepared, a rotor is placed on a magnetic stirrer, the rotation speed is adjusted to 200-220 rpm, and then the copper ion solution is added dropwise.

[0012] Furthermore, after adding the copper ion solution, intermittent ultrasonic treatment was performed for 5 to 10 minutes (ultrasonic treatment for 30 seconds and rest for 10 seconds). After the ultrasonic treatment was completed, the mixture was allowed to stand for 30 to 60 minutes, filtered under reduced pressure, and the residue was taken out and fully washed with deionized water and dried at room temperature.

[0013] Application of in-situ synthesis of immobilized lipase using copper-based metal organic materials in immobilized enzymes The catalytic activities of free lipase and immobilized lipase were detected according to the national standard method (GB / T 23535-2009), and the enzyme activity retention rate before and after immobilization was inferred.

[0014] The Bradford method was used to detect the free enzyme protein content in the filtrate, calculate the loading amount of immobilized lipase, and investigate its immobilization efficiency.

[0015] The relative enzyme activities of free enzyme and immobilized lipase at 20~70℃ were investigated, and their temperature stability was compared.

[0016] The changes in enzymatic activity of immobilized lipase after repeated use were investigated, and its operational stability was examined.

[0017] The present invention is beneficial in that: (1) The present invention uses a metal organic material prepared from copper and 2-aminoterephthalic acid to immobilize lipase. The preparation process can be completed in an aqueous phase at room temperature, and the catalytic activity of the lipase after immobilization is increased by more than 10%.

[0018] (2) The copper ions of the present invention, as a metal source, improve the enzyme activity and form a metal organic material with 2-aminoterephthalic acid, in which the enzyme is encapsulated in situ to prepare a metal organic material immobilized lipase. The obtained immobilized lipase has a loading amount of more than 400 mg / g and can retain 63.5% of the original activity after repeated use for more than 6 times, and has better thermal stability. DETAILED DESCRIPTION

[0019] The following examples are used to further illustrate the present invention, but are not intended to limit the scope of the present invention.

[0020] Example 1 Application of high loading rate and high activity immobilized lipase Copper sulfate and 2-aminoterephthalic acid were added to a 0.05 mol / L sodium acetate buffer solution with a pH of 7-8, respectively, and ultrasonically treated until homogeneous, to obtain a copper ion solution with a concentration of 0.04-0.05 mol / L and an organic ligand solution with a concentration of 0.012-0.015 mol / L.

[0021] Take 100 ml of the above organic ligand solution and add lipase, stir evenly to obtain an organic ligand-lipase solution with an enzyme concentration of 0.09-1.0 g / L, add a rotor, place it on a magnetic stirrer, set the speed to 200-220 rpm, and drop 36 ml of the above copper ion solution. Intermittently ultrasonicate (ultrasonicate for 30 s, stop for 10 s) for 7-8 min, let it stand for 45-60 min, then filter under reduced pressure, wash it three times with deionized water, collect the filtrate, dry the solid at room temperature, weigh it, and obtain immobilized lipase.

[0022] Loading capacity investigation: Take the above filtrate and use Bradford method to determine the concentration of free lipase in it. Subtract the mass of free lipase from the mass of added lipase and divide it by the mass of immobilized lipase to get the loading capacity. The loading capacity of immobilized lipase prepared under this condition is ≥427 mg / g.

[0023] Preparation of substrate solution: Weigh 4 g of polyvinyl alcohol, add 80 ml of water, heat in a boiling water bath to dissolve, cool to room temperature and adjust the volume to 100 ml, filter with double-layer gauze, and take the filtrate. Take 90 ml of the above polyvinyl alcohol solution, add 30 ml of olive oil, and stir with a stirrer for 6 minutes until a milky white emulsion is obtained. Prepare and use immediately.

[0024] Enzyme activity determination: Take two triangular bottles and number them, with bottle A as the blank bottle and bottle B as the sample bottle. Add 4 ml of substrate solution and 5 ml of pH 7.5 0.2 mol / L phosphate buffer to each bottle. Then add 15 ml of 95% ethanol to bottle A, preheat bottles AB at 40°C for 5 min, add 0.02 g of immobilized enzyme to bottle B, mix immediately, react for 15 min, add 15 ml of 95% ethanol to bottle B to stop the reaction, and take it out. Add 2 drops of phenolphthalein indicator solution to each bottle AB, titrate with 0.05 mol / L sodium hydroxide, and calculate the enzyme activity according to the following formula.

[0025] Calculation formula

[0026] Where: X—enzyme activity of the sample, u / g; V 1 —The volume of sodium hydroxide standard solution consumed when titrating the sample, in milliliters (mL); V 2 —The volume of sodium hydroxide standard solution consumed when titrating the blank, in milliliters (mL); c—concentration of sodium hydroxide standard solution, in moles per liter (mol / L); 1.00 mL of 50-0.05 mol / L sodium hydroxide solution is equivalent to 50 μmol of fatty acid; n 1 —Dilution factor of the sample; 0.05—conversion coefficient of sodium hydroxide standard solution concentration; —Reaction time is 15 min, calculated as 1 min.

[0027] Investigation on enzyme activity retention rate: Taking the loading amount of immobilized enzyme as 427 mg / g, take the corresponding mass of immobilized lipase and free lipase, and determine the enzyme activity according to the above steps. The ratio of the two enzyme activities is the enzyme activity retention rate. The activity of the immobilized lipase prepared under this condition increased by more than 14%.

[0028] Example 2 Application of high stability immobilized lipase Copper sulfate and 2-aminoterephthalic acid were added to a 0.05 mol / L sodium acetate buffer solution with a pH of 7-8, respectively, and ultrasonically treated until homogeneous, to obtain a copper ion solution with a concentration of 0.03-0.04 mol / L and an organic ligand solution with a concentration of 0.010-0.012 mol / L.

[0029] Take 100 ml of the above organic ligand solution, add lipase, stir evenly, obtain an organic ligand-lipase solution with an enzyme concentration of 1.1-1.2 g / L, add a rotor, place it on a magnetic stirrer, set the speed to 200-220 rpm, and drop 33 ml of the above copper ion solution. Intermittently ultrasonicate (ultrasonicate for 30 s, stop for 10 s) for 6-8 min, let it stand for 30-60 min, then filter under reduced pressure, take the filter residue, wash it three times with deionized water, and dry it at room temperature to obtain immobilized lipase.

[0030] Preparation of substrate solution: Weigh 4 g of polyvinyl alcohol, add 80 ml of water, heat in a boiling water bath to dissolve, cool to room temperature and adjust the volume to 100 ml, filter with double-layer gauze, and take the filtrate. Take 90 ml of the above polyvinyl alcohol solution, add 30 ml of olive oil, and stir with a stirrer for 6 minutes until a milky white emulsion is obtained. Prepare and use immediately.

[0031] Enzyme activity determination: Take two triangular bottles and number them, with bottle A as the blank bottle and bottle B as the sample bottle. Add 4 ml of substrate solution and 5 ml of pH 7.5 0.2 mol / L phosphate buffer to each bottle. Then add 15 ml of 95% ethanol to bottle A, preheat bottles AB at 40°C for 5 min, add 0.02 g of immobilized lipase to bottle B, mix immediately, react for 15 min, add 15 ml of 95% ethanol to bottle B to stop the reaction, and take it out. Add 2 drops of phenolphthalein indicator solution to each bottle AB, titrate with 0.05 mol / L sodium hydroxide, and calculate the enzyme activity according to the following formula.

[0032] Calculation formula

[0033] Where: X—enzyme activity of the sample, u / g; V 1 —The volume of sodium hydroxide standard solution consumed when titrating the sample, in milliliters (mL); V 2 —The volume of sodium hydroxide standard solution consumed when titrating the blank, in milliliters (mL); c—concentration of sodium hydroxide standard solution, in moles per liter (mol / L); 1.00 mL of 50-0.05 mol / L sodium hydroxide solution is equivalent to 50 μmol of fatty acid; n 1 —Dilution factor of the sample; 0.05—conversion coefficient of sodium hydroxide standard solution concentration; —Reaction time is 15 min, calculated as 1 min.

[0034] Recycling rate investigation: First, adjust the pH of the solution to be hydrolyzed to between 7 and 8 with phosphate buffer, preheat at 30-45°C, add immobilized lipase, and add 4-5 g / L of immobilized lipase. Each batch reacts at 30-45°C for more than 15 min. After the reaction is completed, filter and recover the immobilized lipase. After repeated use for six times, the enzyme activity of the immobilized lipase is determined according to the above steps, and its enzyme activity is still maintained at more than 63.5% of the maximum enzyme activity.

[0035] Thermal stability study: The immobilized lipase was prepared according to the enzyme activity determination steps in this example, and the preheating and reaction temperatures during the enzyme activity determination were set to 20°C, 30°C, 40°C, 50°C, 60°C, and 70°C, respectively, and the enzyme activity at each temperature was determined. The immobilized lipase prepared under this condition has better thermal stability than the free enzyme. At 40°C, the immobilized lipase retains more than 95% of the original activity, while the free enzyme only retains 40% of the original activity.

Claims

1. A method for preparing immobilized lipase by in-situ synthesis of copper-based metal organic materials, characterized in that: The preparation method comprises the following steps: firstly, sodium acetate, glacial acetic acid and distilled water are mixed in a certain proportion, and stirred at room temperature to obtain an acetate buffer solution; secondly, a certain mass of copper salt is added to the acetate buffer solution, and a copper ion solution is obtained by ultrasonic treatment; thirdly, 2-aminoterephthalic acid is added to the acetate buffer solution, and firstly stirred and ultrasonicated to dissolve, and then lipase is added, and stirred to dissolve, and the copper ion solution is added dropwise while stirring, and the copper-based metal organic material immobilized lipase is obtained by suction filtration and drying.

2. The method for preparing immobilized lipase by in-situ synthesis of copper-based metal organic materials according to claim 1, characterized in that: The copper salt is preferably at least one of copper sulfate, copper acetate and copper chloride.

3. The method for preparing immobilized lipase by in-situ synthesis of copper-based metal organic materials according to claim 1, characterized in that: Cu 2+ The ratio with H2ATA is 0.42 / 1~1.17 / 1 g / g.

4. The method for preparing immobilized lipase by in-situ synthesis of copper-based metal organic materials according to claim 1, characterized in that: Cu 2+ The ratio with lipase is 0.55 / 1~2.55 / 1 g / g.

5. The method for preparing immobilized lipase by in-situ synthesis of copper-based metal organic materials according to claim 1, characterized in that: After the copper ion solution was added dropwise, intermittent ultrasound was performed for 5 to 10 min (ultrasound for 30 s and rest for 10 s). After the ultrasound was completed, it was allowed to stand for 30 to 60 min, filtered, and the filter residue was fully washed with deionized water and taken out and dried at room temperature to obtain copper-based metal organic material immobilized lipase.

6. Use of the copper-based metal organic material prepared by the preparation method according to any one of claims 1 to 5 for in-situ synthesis of immobilized lipase in immobilized enzymes.