Cyclodextrin-metal organic framework immobilized lipase and application thereof in grease catalysis
By immobilizing lipases on a metal-organic framework carrier modified with cyclodextrin, the problems of poor stability and low catalytic efficiency of lipases are solved, achieving a highly efficient and reusable lipase catalytic effect, which is suitable for the field of oil catalysis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-18
- Publication Date
- 2026-03-27
AI Technical Summary
In existing technologies, free lipases have poor stability, are easily affected by temperature and pH, and are difficult to recycle and reuse, resulting in high catalytic costs. Single metal-organic framework supports have weak interactions with lipases, are prone to detachment after immobilization, and lack specific recognition ability for substrates, so catalytic efficiency needs to be improved.
A metal-organic framework composite carrier modified with cyclodextrin was used to immobilize lipase through physical adsorption and host-guest inclusion synergy. The preparation process was simple. The host-guest inclusion effect of cyclodextrin enhanced the binding stability of lipase and carrier and improved the recognition and binding ability of oil substrates.
It significantly improves the catalytic performance and reusability of lipase, enhances enzyme activity recovery and long-term stability, has high catalytic efficiency, mild reaction conditions, and easy product separation, making it suitable for industrial applications.
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Figure BDA0005747367980000031 
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological catalysis, and particularly relates to a cyclodextrin-metal organic framework immobilized lipase, a preparation method and application thereof in oil catalysis. BACKGROUND
[0002] Lipase (EC 3.1.1.3) is a kind of hydrolase capable of catalyzing the hydrolysis, ester exchange and esterification of oil and fat (triglyceride), and is widely used in food processing, detergent, biodiesel preparation, fine chemical industry and other fields. However, free lipase has poor stability, is easily inactivated by reaction conditions such as temperature and pH value, and is difficult to recover after reaction, which cannot be reused, resulting in high catalytic cost and limiting its large-scale application in industrial production.
[0003] To solve the above problems, immobilization technology is a key means to improve the stability of lipase and realize its reuse. The selection of immobilization carrier is the core of immobilization technology. Currently, commonly used carriers include inorganic carriers (such as silica gel, activated carbon), organic carriers (such as resin, cellulose) and composite carriers. Among them, metal organic framework (MOFs) as a new type of porous material has the advantages of large specific surface area, high porosity and adjustable structure, and shows good application potential in enzyme immobilization field. However, the interaction between single metal organic framework carrier and lipase is weak, and the immobilized lipase is easy to fall off, and lacks specific recognition ability to substrates, resulting in further improvement of catalytic efficiency.
[0004] Cyclodextrin is a cyclic oligosaccharide formed by D-glucose units connected by α-1,4 glycosidic bonds. Its molecular structure has an outer hydrophilic and inner hydrophobic cavity, which can form a stable complex with biological macromolecules such as lipase through host-guest inclusion, and can enhance the stability of the enzyme. The combination of cyclodextrin and metal organic framework to prepare cyclodextrin modified metal organic framework composite carrier can not only realize the efficient loading of lipase by taking advantage of the high specific surface area and high porosity of metal organic framework, but also enhance the binding stability of lipase and carrier through the host-guest inclusion of cyclodextrin, and improve the recognition and binding ability to oil substrates, thereby significantly improving the catalytic performance of immobilized lipase.
[0005] At present, there are few studies on cyclodextrin-metal organic framework composite carrier immobilized lipase, and the existing technology has the problems of complex carrier modification process, poor catalytic activity and reusability of immobilized lipase. Therefore, it is of great significance to develop a cyclodextrin-metal organic framework immobilized lipase with simple preparation process and excellent catalytic performance for promoting the industrial application of lipase in oil catalysis field. SUMMARY
[0006] The application provides a cyclodextrin-metal organic framework composite carrier immobilized lipase, a preparation method and application thereof.
[0007] The cyclodextrin-metal organic framework immobilized lipase uses a cyclodextrin modified metal organic framework as a carrier, and immobilizes the lipase through physical adsorption and host-guest inclusion synergistic effect.
[0008] Further preferably, the cyclodextrin is grafted to the surface of the metal organic framework through a covalent bond, and the cyclodextrin is one or a mixture of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin.
[0009] Further preferably, the metal organic framework is one or a mixture of Zn-MOF, Cu-MOF and Fe-MOF.
[0010] Further preferably, the mass ratio of the lipase to the carrier is 1:5 to 1:20.
[0011] Further preferably, the raw materials for preparing the metal organic framework include a metal salt and an organic ligand; the metal salt is one or a mixture of zinc nitrate, copper chloride and iron chloride; the organic ligand is one or a mixture of 2-methylimidazole, terephthalic acid and trimesic acid; and the molar ratio of the metal salt to the organic ligand is 1:2 to 1:5.
[0012] Further, the application further provides a preparation method of the cyclodextrin-metal organic framework immobilized lipase.
[0013] (1) the metal salt and the organic ligand are dissolved in a solvent, stirred uniformly, transferred to a reaction kettle, reacted at 80-120 DEG C for 12-24 hours, cooled to room temperature, centrifuged, washed and dried to obtain the metal organic framework;
[0014] (2) the metal organic framework obtained in step (1) is dispersed in a solvent, cyclodextrin and a coupling agent are added, reacted at 30-60 DEG C for 4-12 hours, centrifuged, washed and dried to obtain the cyclodextrin modified metal organic framework;
[0015] (3) the cyclodextrin modified metal organic framework obtained in step (2) is dispersed in a phosphate buffer solution, the lipase is added, oscillated and adsorbed at 25-40 DEG C for 6-24 hours; the precipitate is collected by centrifugation, washed with the phosphate buffer solution for 2-3 times, and vacuum dried to obtain the cyclodextrin-metal organic framework immobilized lipase.
[0016] Further preferably, the coupling agent in step (2) is a mixture of 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS), the molar ratio of EDC to NHS is 1:1-1:1.5, and the mass ratio of cyclodextrin to metal-organic framework is 1:10-1:30.
[0017] Further preferably, the lipase in step (3) is one or more of Candida antarctica lipase CalA, CalB, Thermomyces lanuginosus lipase TL, TM, Rhizomucor miehei lipase RM, and phospholipase.
[0018] The application also provides a use of the cyclodextrin-metal-organic framework immobilized lipase described above in catalysis of oils and fats, including but not limited to hydrolysis of oils and fats, transesterification of oils and fats, and esterification of oils and fats. DETAILED DESCRIPTION
[0019] The application is further described in conjunction with specific examples, which can help those skilled in the art to better understand and master the application, but not limit the application. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art.
[0020] Example 1: Preparation of immobilized lipase
[0021] 1. Preparation of metal-organic framework (Zn-MOF carrier)
[0022] 2.97 g of zinc nitrate and 2.46 g of 2-methylimidazole were weighed and dissolved in 50 mL of deionized water, respectively, and the 2-methylimidazole solution was slowly added to the zinc nitrate solution, stirred for 30 min, and then transferred to a reaction kettle for reaction at 100℃ for 20 h. After cooling to room temperature, the precipitate was collected by centrifugation and washed with deionized water and ethanol alternately for 3 times, and then dried under vacuum at 60℃ for 12 h to obtain Zn-MOF.
[0023] 2. Preparation of cyclodextrin-modified Zn-MOF carrier
[0024] 1.0 g of Zn-MOF was dispersed in 50 mL of N,N-dimethylformamide, and 0.2 g of β-cyclodextrin, 0.1 g of EDC and 0.08 g of NHS were added, and the mixture was stirred and reacted at 50℃ for 8 h. The precipitate was collected by centrifugation and washed with N,N-dimethylformamide and deionized water alternately for 3 times, and then dried under vacuum at 60℃ for 12 h to obtain a β-cyclodextrin-modified Zn-MOF carrier.
[0025] 3. Preparation of cyclodextrin-metal-organic framework immobilized lipase
[0026] Take 0.2 g of the β-cyclodextrin modified Zn-MOF carrier, disperse it in 20 mL of a phosphate buffer solution with a pH of 7.0, add 0.02 g of lipase RM, and oscillate for adsorption at 30°C for 12 h; collect the precipitate by centrifugation, wash it with a phosphate buffer solution with a pH of 7.0 for 3 times, and vacuum dry it at 40°C for 8 h to obtain the cyclodextrin-metal organic framework immobilized lipase.
[0027] 4. Preparation of a metal organic framework immobilized lipase without cyclodextrin modification
[0028] Take 0.2 g of the Zn-MOF carrier, disperse it in 20 mL of a phosphate buffer solution with a pH of 7.0, add 0.02 g of lipase RM, and oscillate for adsorption at 30°C for 12 h; collect the precipitate by centrifugation, wash it with a phosphate buffer solution with a pH of 7.0 for 3 times, and vacuum dry it at 40°C for 8 h to obtain the metal organic framework immobilized lipase without cyclodextrin modification.
[0029] Take the cyclodextrin-metal organic framework immobilized lipase prepared in Example 1, the commercial HPD-600 macroporous resin immobilized Palatase 20000L lipase (denoted as sample B, purchased from a biological technology company, with an enzyme activity marked as 1400 U / g), and the commercial silanized diatomite immobilized lipase (denoted as sample C, purchased from a biological technology company, with an enzyme activity marked as 1200 U / g) respectively, and determine the key enzyme parameters under the same test conditions (pH 7.0, temperature 45°C, and olive oil as the substrate).
[0030] Table 1. Enzyme parameter results of the cyclodextrin-metal organic framework immobilized lipase and the commercial immobilized lipases
[0031]
[0032]
[0033] As can be seen from Table 1, the enzyme activity recovery rate of the cyclodextrin-metal organic framework immobilized lipase (sample A) prepared in the application is significantly higher than that of sample B, and although it is slightly lower than that of sample C, it has obvious advantages in long-term repeated use: after being repeatedly used for 8 times, the relative enzyme activity of sample A still remains above 60%, while that of sample B and sample C respectively decreases to below 40% and below 55%.
[0034] Example 3. Application of the cyclodextrin-metal organic framework immobilized lipase in oil hydrolysis reaction
[0035] Take 5 g of soybean oil into a 50 mL three-necked flask, add 20 mL of phosphate buffer solution with pH 7.5, and then add 0.1 g of each of the two immobilized lipases prepared in Example 1 and the commercial immobilized lipases (Sample B and Sample C), respectively, and stir the reaction at 45°C for 6 h; after the reaction is completed, the amount of fatty acid generated is determined by titration, and the oil hydrolysis rate is calculated.
[0036] The results show that the oil hydrolysis rate using the cyclodextrin-metal organic framework immobilized lipase is more than 85%, and after the immobilized lipase is recovered and reused for 6 times, the oil hydrolysis rate still remains more than 65%; the oil hydrolysis rate using the metal organic framework immobilized lipase without cyclodextrin modification is 65%, and after the immobilized lipase is reused for 3 times, the oil hydrolysis rate decreases to less than 40%; the hydrolysis rate of Sample B is 68%-72%, and the hydrolysis rate of Sample C is 82%-85%. It is indicated that the cyclodextrin-metal organic framework immobilized lipase prepared in the application has higher catalytic activity and better reusability.
[0037] Example 4 Application of the cyclodextrin-metal organic framework immobilized lipase in oil transesterification reaction
[0038] Take 10 g of rapeseed oil into a 50 mL three-necked flask, add 3.6 g of methanol (the molar ratio of oil to methanol is 1:6), and then add 0.5 g of each of the cyclodextrin-metal organic framework immobilized lipase prepared in Example 1 and the commercial immobilized lipases (Sample B and Sample C), and stir the reaction at 45°C for 12 h; after the reaction is completed, the immobilized lipase is separated by centrifugation, and the yield of biodiesel (fatty acid methyl ester) is determined by gas chromatography. The results show that the yield of biodiesel using the cyclodextrin-metal organic framework immobilized lipase is more than 90%; after the immobilized lipase is recovered and reused for 5 times, the yield of biodiesel still remains more than 70%; the yield of Sample B is 75%-80%, and the yield of Sample C is 85%-88%.
[0039] In addition, the cyclodextrin-metal organic framework immobilized lipase provided in the application has a clear porous structure and good mechanical stability, and can be efficiently separated by simple centrifugation after the reaction, with a separation efficiency of more than 95%; Sample B (macroporous resin carrier) is prone to slight swelling in the reaction system, and needs an additional washing step after separation, with a separation efficiency of about 85%; Sample C (silica carrier) is convenient to separate, but a small amount of fine particles is easily left, which affects the purity of the product, and a filtration step needs to be added.
[0040] Therefore, the cyclodextrin-metal organic framework immobilized lipase provided by the application has obvious comprehensive advantages in catalytic efficiency, long-term reusability and product separation convenience compared with commercial macroporous resin and silica carrier immobilized lipases, and is more suitable for industrial large-scale application.
[0041] In conclusion, the cyclodextrin-metal organic framework immobilized lipase provided by the application has the advantages of simple preparation process, controllable cost, high catalytic efficiency, good stability and strong reusability; when applied to oil catalytic reaction, the reaction condition is mild, the product is easy to separate, and the cyclodextrin-metal organic framework immobilized lipase can be widely applied to food processing, biodiesel preparation, fine chemical industry and other industrial fields, and has significant economic value and social benefits.
Claims
1. A cyclodextrin-metal organic framework immobilized lipase, characterized in that, The immobilized lipase is carried by a cyclodextrin-modified metal organic framework, and is fixed with the lipase through physical adsorption.
2. The cyclodextrin-metal-organic framework immobilized lipase of claim 1, wherein, The cyclodextrin is one or more of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin.
3. The cyclodextrin-metal-organic framework immobilized lipase of claim 1, wherein, The metal organic framework is one or more of Zn-MOF, Cu-MOF and Fe-MOF.
4. A method of preparing a cyclodextrin-metal organic framework immobilized lipase, characterized by, The method comprises the following steps: (1) dissolving a metal salt and an organic ligand in a solvent, stirring uniformly, transferring to a reaction kettle, reacting at 80-120°C for 12-24h, cooling to room temperature, centrifuging, washing and drying to obtain a metal organic framework; (2) dispersing the metal organic framework obtained in step (1) in a solvent, adding cyclodextrin and a coupling agent, reacting at 30-60°C for 4-12h, centrifuging, washing and drying to obtain a cyclodextrin-modified metal organic framework; (3) dispersing the cyclodextrin-modified metal organic framework obtained in step (2) in a phosphate buffer solution, adding a lipase, oscillating and adsorbing at 25-40°C for 6-24h; centrifuging to collect the precipitate, washing with a phosphate buffer solution for 2-3 times, vacuum drying to obtain a cyclodextrin-metal organic framework immobilized lipase.
5. The preparation method of cyclodextrin-metal organic framework immobilized lipase according to claim 4, characterized in that, The coupling agent in step (2) is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), the molar ratio of EDC to NHS is 1:1-1:1.5, and the mass ratio of cyclodextrin to metal organic framework is 1:10-1:
30.
6. The method of claim 4, wherein, The lipase in step (3) is one or more of Candida antarctica lipase CalA, CalB, Thermomyces lanuginosus lipase TL, TM, Rhizomucor miehei lipase RM and phospholipase.
7. The cyclodextrin-metal organic framework immobilized lipase according to claim 1 in the catalysis of oil.