Immobilized lipase for preparing biodiesel as well as preparation method and application of immobilized lipase

Abstract

The invention discloses immobilized lipase for preparing biodiesel as well as a preparation method and application of the immobilized lipase. The immobilized lipase comprises a metal organic framework carrier modified by a hydrophobic group and lipase immobilized on the metal organic framework carrier, the hydrophobic group is selected from n-octadecyl phosphate (OPA); the metal organic framework is UiO-66-NH2, and the structural formula of the metal organic framework is as follows; the immobilized lipase can also be combined with a nonionic surfactant to be used as a co-catalyst; the temperature tolerance, the pH tolerance and the storage time tolerance of the immobilized lipase are respectively 13.66%, 39.99% and 17.10% higher than those of free enzymes; the compound shows relatively strong tolerance to Fe < 2 + > and Fe < 3 + >, which are 3.27 times and 4.07 times of those of free enzyme respectively, and the catalytic efficiency is also effectively improved after a nonionic surfactant is added.

Description

Technical Field

[0001] The present invention relates to the technical field of biodiesel preparation, and in particular to an immobilized lipase for preparing biodiesel, a preparation method and an application thereof. Background Art

[0002] In recent decades, due to the depletion of fuel resources and their impact on the environment, biodiesel has emerged as a sustainable alternative to fossil fuels. Biodiesel refers to fatty acid methyl or ethyl esters formed by the transesterification of vegetable oils (such as sunflower oil, rapeseed oil, soybean oil, peanut oil, corn oil, and cottonseed oil), animal oils (such as fish oil, lard, tallow, and mutton oil), waste oils, or microbial oils with alcohols. This renewable energy source exhibits favorable combustion characteristics, including reduced CO emissions, the absence of sulfur compounds, low aromatic hydrocarbon content, an elevated cetane number, and enhanced flash point stability.

[0003] Biodiesel production technology is mainly based on chemical catalysis and enzymatic catalysis. The application of the former is gradually restricted due to its high corrosiveness, high energy consumption and high pollution. Lipase (Lipase, LIP) is a class of hydrolases that can hydrolyze triglycerides into glycerol and fatty acids. It is widely present in animals, plants and microorganisms. Its action depends on the oil-water interface and its optimal pH is alkaline. It has the characteristics of catalyzing ester synthesis and transesterification and has significant potential in the field of biodiesel preparation. Although the biocatalytic method using lipase has operational advantages, its scalability has been challenged due to its fragility under non-ideal process conditions, limited operating life and inefficient recovery schemes. Therefore, the design of a robust immobilized lipase enzyme system with higher stability and reusability has become a research focus.

[0004] Nonionic surfactants are amphiphilic compounds composed of hydrophilic and hydrophobic groups. They can modify interfacial properties by reducing liquid surface tension and forming micelles, and are widely used in the cosmetic, industrial, and biological fields. Nonionic surfactants of the same type and concentration have differential effects on lipase activity, inducing exposure of the enzyme's active center and stabilizing the open conformation of the catalytic "lid" through interfacial activation. This process reduces local water activity, pH fluctuations, and ionic strength gradients, while also inhibiting the adverse effects of organic solvents or extreme environmental conditions on enzyme catalytic activity. However, optimization technologies for biodiesel biocatalysis using nonionic surfactants as media remain underdeveloped.

[0005] UiO-66-NH2 is a metal-organic framework (MOF) material with a highly ordered nanoscale pore structure and tunable amino functional groups. It can efficiently load lipase through physical adsorption or chemical bonding, improving the enzyme's immobilization efficiency and becoming a mainstream immobilization carrier for lipase. Despite these advantages, the interfacial activation synergistic catalysis of nonionic surfactants and immobilized enzymes in biodiesel production is still lacking.

[0006] CN 114540334 A discloses a method for preparing biodiesel from oil by catalyzing lipase loaded on a metal organic framework material. The method comprises subjecting the metal organic framework material to hydrophobic treatment, loading the material with lipase after the hydrophobic treatment, and using the material to catalyze the preparation of biodiesel. However, the method has problems such as the excessively high temperature used during the hydrophobic modification process, which easily destroys the crystal structure of the MOF, resulting in a decrease in the catalytic efficiency of the immobilized enzyme, and the complex synthesis method. In response to this problem, an immobilized lipase for preparing biodiesel, a preparation method, and an application thereof are proposed. Summary of the Invention

[0007] To address these technical issues, the present invention proposes an immobilized lipase for biodiesel production, a preparation method, and its application. This method is simple to operate, safe, environmentally friendly, and significantly improves catalytic activity, demonstrating its universal applicability. By hydrophobically modifying UiO-66-NH2, the present invention designed an immobilized support (OPA / UiO-66-NH2) on which lipase (LIP) was immobilized. This immobilized support, combined with a nonionic surfactant, is used for the first time to catalyze biodiesel production. Comprehensive evaluation of key reaction parameters demonstrates high catalytic activity, stability, and reusability, providing a novel approach for biodiesel production.

[0008] According to a first aspect of the present invention, there is provided an immobilized lipase for preparing biodiesel, wherein the immobilized lipase comprises a metal organic framework carrier modified with hydrophobic groups and a lipase immobilized on the metal organic framework carrier;

[0009] The lipase immobilized on the metal organic framework support can be used in combination with a nonionic surfactant as a co-catalyst;

[0010] The hydrophobic group is selected from OPA.

[0011] Optionally, the metal organic framework is selected from at least one of the ZIF series, MIL series, PCN series, and UiO series.

[0012] Optionally, the metal organic framework is of the UiO series.

[0013] Optionally, the metal organic framework is UiO-66-NH2.

[0014] Optionally, the metal organic framework is UiO-66-NH2, the hydrophobic group is OPA, wherein the mass ratio of UiO-66-NH2 to OPA is 50:1~5, and OPA is covalently bonded to the surface of UiO-66-NH2 to obtain a metal organic framework carrier.

[0015] Optionally, the mass ratio of the lipase to the metal organic framework carrier is 1-5:5-10;

[0016] The nonionic surfactant is selected from at least one of fatty acid methyl ester ethoxylate, fatty alcohol polyoxyethylene ether, polyoxyethylene lauryl alcohol, Span-80, and Tween-80;

[0017] The mass concentration of the nonionic surfactant is 0.5-2%;

[0018] The mass ratio of the nonionic surfactant to the immobilized enzyme is 1-4:6-48.

[0019] According to a second aspect of the present invention, a method for preparing immobilized lipase is provided, the method comprising at least the following steps:

[0020] Step 1, synthesis of UiO-66-NH2: zirconium chloride and 2-aminoterephthalic acid are dissolved in N,N-dimethylformamide, ultrasonically dissolved, acetic acid and water are added, stirred, then N,N-dimethylformamide is added, stirred, and transferred to a hydrothermal reactor for reaction to obtain an orange-red suspension. The product is collected by centrifugation, washed with N,N-dimethylformamide and methanol, and vacuum dried to obtain UiO-66-NH2;

[0021] Step 2, synthesis of OPA / UiO-66-NH2: dissolve OPA in ethanol, disperse by ultrasonication, add UiO-66-NH2 obtained in step 1, react at room temperature for 24 to 48 hours, stirring for 5 minutes every 2 hours, wash the reaction product by centrifugation with ethanol, and dry in vacuo to obtain OPA / UiO-66-NH2;

[0022] Step 3, immobilization of lipase: ultrasonically disperse the OPA / UiO-66-NH2 obtained in step 2 into isopropanol, add PBS solution containing lipase, shake well and place on a shaker, react, centrifuge, wash, and freeze-dry to obtain immobilized lipase.

[0023] Optionally, in step 2, the mass mixing ratio of OPA to UiO-66-NH2 is 50:1-5.

[0024] The reaction conditions in step 3 are as follows:

[0025] The reaction time is 0.5 to 1 h;

[0026] The reaction temperature is 30 to 50 degrees Celsius;

[0027] The reaction was carried out at a speed of 150 to 180 rpm;

[0028] The concentration of the lipase in the PBS solution containing lipase in step 3 is 2-5 mg / mL.

[0029] According to a third aspect of the present invention, there is provided an application of an immobilized lipase in the preparation of biodiesel, wherein the immobilized lipase is mixed with an oil substrate, a co-solvent is added, preheated, an organic alcohol is added, and an ester exchange reaction is carried out. After the reaction is completed, the immobilized lipase is centrifuged and the biodiesel is rotary evaporated.

[0030] The oil substrate is selected from at least one of sunflower oil, rapeseed oil, and olive oil;

[0031] The co-solvent is selected from at least one of n-hexane, toluene, and tetrahydrofuran;

[0032] The mass mixing ratio of the immobilized enzyme to the oil substrate is 15-120:264.

[0033] Optionally, the preheating temperature is 35 to 45 degrees Celsius;

[0034] The organic alcohol is selected from at least one of methanol, ethanol and butanol;

[0035] The volume ratio of the organic alcohol to the oil substrate is 1 to 5:1;

[0036] The conditions for the transesterification reaction of the organic alcohol and the ester exchange reaction are as follows:

[0037] The transesterification reaction time is 12 to 72 hours;

[0038] The temperature of the transesterification reaction is 30 to 50 degrees Celsius.

[0039] Optionally, a nonionic surfactant is added before the transesterification reaction;

[0040] The nonionic surfactant is selected from at least one of fatty acid methyl ester ethoxylate, fatty alcohol polyoxyethylene ether, polyoxyethylene lauryl alcohol, Span-80, and Tween-80;

[0041] The mass concentration of the nonionic surfactant is 0.5-2%;

[0042] The mass ratio of the nonionic surfactant to the immobilized enzyme is 1-4:6-48.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] (1) The technical solution adopted by the present invention is to hydrophobically modify the metal organic framework through OPA. This method greatly improves the catalytic activity of the immobilized lipase (167.39%). The temperature, pH and storage time tolerance are 13.66%, 39.99% and 17.10% higher than those of the free enzyme respectively; 2+ and Fe 3+ They showed strong tolerance, which was 3.27 and 4.07 times that of the free enzyme, respectively.

[0045] (2) The technical solution adopted by the present invention also added a non-ionic surfactant (i.e., 2% FMEE, 2% AEO9, and 2% Brji) to the catalytic system, and it was found that the catalytic efficiency was increased by 14.39%, 5.29%, and 0.57%, respectively. The non-ionic surfactant is an amphiphilic compound composed of a hydrophilic group and a hydrophobic group, which can disperse the originally stratified reactants into tiny droplets under the emulsification effect. The formed droplets can increase the oil-water interface and play a co-catalytic effect, thereby improving the catalytic activity.

[0046] (3) The immobilized lipase obtained by the technical solution adopted in the present invention has a high cycle efficiency. Compared with the free enzyme, the catalytic efficiency decreased by 61.33% after 7 cycles, while that of OPA / UiO-66-NH2@LIP only decreased by 3.09%. On this basis, after adding 2% FMEE, 99.15% of fatty acid methyl esters (FAME) were successfully converted in the first catalysis. After 11 cycles, the catalytic efficiency still reached 82.67%. Since lipase relies on the oil-water interface to exert its catalytic effect, exposing active sites and enhancing its ability to bind to the substrate, but lipase is easily inactivated under external conditions, the immobilization of the material can greatly improve its stability and protect it, thereby achieving efficient catalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Scanning electron microscope images of UiO-66-NH2, UiO-66-NH2@LIP, OPA / UiO-66-NH2, and OPA / UiO-66-NH2@LIP obtained in Example 1 of the present invention (where a is UiO-66-NH2, b is UiO-66-NH2@LIP, c is OPA / UiO-66-NH2, and d is OPA / UiO-66-NH2@LIP);

[0048] Figure 2 The test results of enzyme loading and relative activity of immobilized lipase after hydrophobic modification with different concentrations of OPA in Example 2 of the present invention are as follows;

[0049] Figure 3The stability test results of OPA / UiO-66-NH2@LIP in Example 3 of the present invention are shown (where a represents thermal stability, b represents pH stability, c represents storage stability, d represents metal ions, e represents organic solvents, and f represents high-concentration surfactants);

[0050] Figure 4 The test results of biodiesel yield using different transesterification reaction conditions in Example 4 of the present invention (where a is the transesterification reaction time, b is the transesterification reaction temperature, c is the amount of OPA / UiO-66-NH2@LIP added, and d is the molar ratio of methanol to oil substrate);

[0051] Figure 5 The test results of Example 5 of the present invention using different surfactants for the catalytic efficiency of biodiesel (where I is Free LIP, II is AEO9, III is FMEE, IV is Brji, V is Span 80, and VI is Tween 80); Figure 6 These are the reusability test results of Free LIP, OPA / UiO-66-NH2@LIP, and OPA / UiO-66-NH2@LIP+2%FMEE in Example 6 of the present invention. DETAILED DESCRIPTION

[0052] The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto in any way.

[0053] Phosphate buffer saline (PBS, pH = 7.50 ± 0.50) was prepared according to the national standard (GB / T 42239.2-2022) as follows: 3.92 g of potassium dihydrogen phosphate and 79.24 g of disodium hydrogen phosphate dodecahydrate were weighed, approximately 450 mL of water were added, and the mixture was stirred until uniformly dissolved. The pH was adjusted to 7.50 ± 0.05 with 0.5 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid solution, and the volume was adjusted to 500 mL to obtain a PBS solution with a concentration of 0.1 mol / L.

[0054] The immobilization of lipase was tested using a Sigma-360 scanning electron microscope from Zeiss, Germany.

[0055] The lipase (LIP) used in the examples was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (catalog number: L874984-25g, CAS: 9001-62-1, 20000 U / g); the oil substrate used in the examples, i.e., sunflower oil, was purchased from COFCO Fortune Food Marketing Co., Ltd.

[0056] The nonionic surfactants used in the examples, fatty alcohol polyoxyethylene ether (AEO9), fatty acid methyl ester ethoxylate (FMEE), polyoxyethylene lauryl alcohol (Brji), Span-80, and Tween-80 were all purchased;

[0057] The MOFs material in Example 7 was prepared by the following method: ZIF-8 (Zn) was prepared by 2+ Ions; ZIF-67(Co) was prepared by the method disclosed in High-performance overall water splitting electrocatalysts derived from cobalt-based metal-organic frameworks; MIL-53(Al) was prepared by the method disclosed in Arationale for the large breathing of the porous aluminum terephthalate(MIL-53)upon hydration; MIL-88A(Fe) was prepared by the method disclosed in Flexible Humidity Sensitive Fiber with Swellable Metal-Organic Frameworks; MIL-101(Fe) was prepared by the method disclosed in Solvent-free allylic oxidation of alkenes with O2mediated by Fe-and Cr-MIL-101; PCN-224(Cr) was prepared by the method disclosed in Size-controlled synthesis of porphyrinic metal-organic framework and functionalization for targeted photodynamic therapy.

[0058] Lipase activity determination method in the embodiment:

[0059] Mix Solution A (30 mmol / L 4-nitrophenyl palmitate (4-NPP)) and Solution B (0.1 mol / L PBS) at a volume ratio of 1:9. Add 2 mL of 1% Triton X-100 to prepare the mixture. Pipette 2.7 mL of this mixture into Tube 1 (blank), Tube 2, Tube 3, and Tube 4. Add 1.00 mL of the pre-inactivated lipase solution to Tube 1, and 1.00 mL of the uninactivated lipase solution to Tubes 2, 3, and 4. Incubate at 37°C for 10 minutes, then terminate the reaction with 1.00 mL of anhydrous ethanol. Using Tube 1 as the blank, measure the absorbance (OD 410 nm) of Tubes 2, 3, and 4 using a 10 mm cuvette at 410 nm. A 0.04-0.2 mmol / L 4-NPP solution was prepared, and its absorbance at a wavelength of 410 nm was measured to obtain a concentration standard curve, and the concentration of the substrate 4-NP obtained by the enzyme-catalyzed reaction was calculated.

[0060] The enzyme activity of the sample was calculated according to formula 1:

[0061]

[0062] In formula 1, X1 is the enzyme activity of sample, U / g;

[0063] c1 Calculate the concentration of p-nitrophenol from the standard curve, μg / mL;

[0064] V1 is the volume of the reaction solution, mL;

[0065] t is the reaction time, min.

[0066] Method for determining enzyme loading in the examples: The Bradford method (Coomassie Brilliant Blue method) is a protein quantification method based on dye binding, which is used to determine the initial enzyme concentration, supernatant enzyme concentration, and wash liquid enzyme concentration (i.e., enzyme loading), and the enzyme loading is calculated by Formula 2:

[0067]

[0068] In formula 2, m1 is the amount of bound protein, mg;

[0069] m2Amount of unbound protein, mg.

[0070] The yield of biodiesel in the examples was measured by gas chromatography internal standard method. The catalytic product was measured in a certain ratio using n-hexane as solvent and methyl nonadecanoate as internal standard, and the yield (%) of biodiesel was calculated by formula 3. The biodiesel (FAME) content in the product was determined using Shimadzu GC 2010pro gas chromatograph. The detector was FID, and the chromatographic conditions were as follows: chromatographic column model AC-20 (30m×0.32mm×0.25μm); the column temperature was programmed: the initial temperature was 160°C for 3 minutes, then increased to 240°C at 10°C / min and held for 29 minutes; the temperature of the injection port and detector were both 300°C; the split ratio was 10:1; and the injection volume was 1.0μL.

[0071]

[0072] In formula 3, wt% biodiesel yield, %;

[0073] A MH Total peak area of ​​fatty acid methyl esters and peak area of ​​methyl nonadecanoate, cm 2 ;

[0074] C MH concentration of methyl nonadecanoate, mg / mL;

[0075] V MH Volume of methyl nonadecanoate, mL;

[0076] W product mass, g.

[0077] Unless otherwise specified, all reagents used in the following examples were purchased.

[0078] Example 1

[0079] (1) Preparation of MOFs material (UiO-66-NH2):

[0080] 0.20g of zirconium chloride and 0.26g of 2-aminoterephthalic acid were weighed and dissolved in 30mL of N,N-dimethylformamide under ultrasonication. 10mL of acetic acid and 4mL of water were added sequentially to the solution, followed by stirring for 5 hours. 10mL of N,N-dimethylformamide was added again, and stirring continued for 5 hours. The solution was transferred to a polytetrafluoroethylene-lined hydrothermal autoclave and reacted at 120°C for 24 hours. Upon completion of the reaction, an orange-red suspension was obtained. The product was collected by centrifugation, washed three times with DMF and three times with methanol, and dried under vacuum at 70°C for 12 hours to obtain UiO-66-NH2.

[0081] (2) Hydrophobic modification of MOFs materials:

[0082] Taking the hydrophobic modification of UiO-66-NH2 with 10% octadecylphosphonic acid (OPA) to produce a multi-stage hydrophobic OPA / UiO-66-NH2 as an example, 0.05g of OPA was weighed and dissolved in 20mL of ethanol under ultrasonication, followed by the addition of 0.5g of UiO-66-NH2. The reaction was allowed to proceed at room temperature for 24 hours, with stirring for 5 minutes every 2 hours. The reactants were washed three times with anhydrous ethanol by centrifugation and finally dried under vacuum at 50°C for 12 hours to obtain 10% OPA / UiO-66-NH2.

[0083] (3) Immobilization of lipase:

[0084] Weigh 0.05 g of 10% OPA / UiO-66-NH2 and ultrasonically disperse it in 1 mL of isopropanol. Add 4 mL of a PBS mixed solution containing 5 mg / mL lipase (LIP) (5 mg of lipase is dissolved in 1 ml of 0.1 mol / L PBS to form a 5 mg / mL lipase (LIP) PBS mixed solution). After fixing the shaker at a constant temperature, take it out and centrifuge it. Take the supernatant for later use to obtain the centrifuged product (OPA / UiO-66-NH2+LIP).

[0085] Weigh 0.05 g of UiO-66-NH2 and ultrasonically disperse it in 1 mL of isopropanol. Add 4 mL of PBS mixed solution containing lipase (LIP) with a concentration of 5 mg / mL. After fixing the shaker at a constant temperature, take it out and centrifuge it. Take the supernatant for later use to obtain the centrifuged product (UiO-66-NH2+LIP).

[0086] The above centrifuged products were washed with PBS solution (0.1 mol / L) until no protein remained in the supernatant, and then freeze-dried for 12 h to obtain immobilized enzymes (OPA / UiO-66-NH2@LIP, UiO-66-NH2@LIP). The scanning electron microscopy test results of UiO-66-NH2, UiO-66-NH2@LIP, OPA / UiO-66-NH2, and OPA / UiO-66-NH2@LIP are shown in Figure 2. Figure 1 As shown, the results indicate that the morphology of OPA / UiO-66-NH2 was preserved after enzyme immobilization and was not significantly affected during the immobilization process.

[0087] (4) Preparation of biodiesel:

[0088] OPA / UiO-66-NH2@LIP was used as a catalyst for the transesterification of methanol and an oil substrate (triglycerides, i.e., sunflower oil) to produce biodiesel. 0.528 g of the oil substrate (triglycerides, i.e., sunflower oil), 180 mg of OPA / UiO-66-NH2@LIP (immobilized enzyme), and 1 mL of a 2% nonionic surfactant (FMEE, fatty acid methyl ester ethoxylate) were weighed into a reaction flask. 3 mL of n-hexane was added as a co-solvent. The flask was preheated at 40°C on a shaker for 5 minutes, followed by the addition of 2.0 mL of methanol (methanol / oil substrate molar ratio of 3:1) in three dropwise portions. The transesterification reaction was carried out at 40°C and a rotation speed of 180 rpm for 48 hours. After termination of the reaction, the immobilized catalyst was separated by centrifugation, and the co-solvent and methanol were removed by rotary evaporation to obtain biodiesel (i.e., fatty acid methyl esters) with a yield of 99.15%.

[0089] Example 2

[0090] The difference from Example 1 is that the hydrophobic modification of the MOF material is to use octadecylphosphonic acid (OPA) with a mass concentration of 2%, 4%, and 6% to hydrophobically modify UiO-66-NH2 to obtain multi-level hydrophobic OPA / UiO-66-NH2.

[0091] Specifically, 0.01g, 0.02g, and 0.03g of OPA were weighed, added to 20mL of ethanol, and ultrasonically dissolved, followed by the addition of 0.5g of UiO-66-NH2. The mixture was reacted at room temperature for 24 hours, with stirring for 5 minutes every 2 hours. The reactants were washed three times with anhydrous ethanol by centrifugation and finally dried under vacuum at 50°C for 12 hours to obtain 2% OPA / UiO-66-NH2, 4% OPA / UiO-66-NH2, and 6% OPA / UiO-66-NH2.

[0092] The obtained 2% OPA / UiO-66-NH2, 4% OPA / UiO-66-NH2, and 6% OPA / UiO-66-NH2 were used to immobilize lipase. The results of the effects of immobilization on enzyme loading and lipase relative activity were as follows: Figure 2 As shown, the results show that the hydrophobicity of UiO-66-NH2 increases with the mass concentration of octadecyl phosphate (OPA). After immobilization of lipase, it was found that the relative enzyme activity also increased with the increase of hydrophobicity. At 10% OPA modification (10% OPA / UiO-66-NH2), the enzyme loading and relative lipase activity reached a maximum of 50.16 mg / g and 167.39%, respectively.

[0093] Example 3

[0094] The stability of lipase (LIP) after immobilization was investigated using 10% OPA / UiO-66-NH2. The stability test results are shown in Figure 2. Figure 3 As shown (where a is thermal stability, b is pH stability, c is storage stability, d is metal ions, e is organic solvent, and f is high concentration surfactant).

[0095] Thermal stability test: The immobilized enzyme (i.e., OPA / UiO-66-NH2@LIP) and the free enzyme (i.e., Free LIP) were dispersed in a pH 7.5 buffer and incubated at 4°C, 20°C, 30°C, 40°C, 50°C, and 60°C for 2 h, respectively. Their relative activities were measured. The thermal stability test results, shown in 3a, demonstrate that at 4°C, the relative activities of both the free and immobilized enzymes were greater than 79%, and the activity of the free enzyme was higher than that after immobilization. Within the range of 20°C to 60°C, OPA / UiO-66-NH2@LIP consistently maintained a higher relative activity than the free enzyme.

[0096] pH stability: The immobilized enzyme (i.e., OPA / UiO-66-NH2@LIP) and the free enzyme (i.e., Free LIP) were dispersed in PBS at pH 5.5, 6.5, 7.5, 8.5, and 9.5, respectively, and incubated at 40°C for 2 h. The relative activities were measured. The pH stability test results are shown in 3b (enzyme activity stability of the free and immobilized enzymes in the pH range of 5.5-9.5). The results show that the relative activity of OPA / UiO-66-NH2@LIP is consistently higher than that of the free enzyme in the pH range of 6.5-9.5, and its relative activity reaches 104.55% at pH 7.5, indicating that OPA / UiO-66-NH2@LIP is more suitable for storage under neutral or weakly alkaline conditions. Storage stability.

[0097] Storage Stability: The immobilized enzyme (i.e., OPA / UiO-66-NH2@LIP) and the free enzyme (i.e., Free LIP) were stored at 4°C, and enzyme activity was measured every other day. The storage stability test results, shown in Figure 3c, show that the relative activity of OPA / UiO-66-NH2@LIP decreased more slowly with prolonged storage compared to the free enzyme (c). After 30 days of storage at 4°C, the free enzyme retained 60% of its original activity, while the immobilized enzyme maintained 77.1% of its original activity, demonstrating a significant increase in relative activity.

[0098] Metal ion test: 13.6 mg ZnCl2, 11.1 mg CaCl2, 13.4 mg CuCl2, 12.6 mg MnCl2, 12.6 mg FeCl2, and 16.2 mg FeCl3 were dissolved in 10 mL of water to form 10 mmoL / L ZnCl2, CaCl2, CuCl2, MnCl2, FeCl2, and FeCl3 solutions, respectively. The immobilized enzyme (i.e., OPA / UiO-66-NH2@LIP) and the free enzyme (i.e., Free LIP) were placed in the above metal solutions and incubated at 40°C for 2 h. The enzyme activity was tested. The test results are shown in Figure 2. Figure 3 As shown in d, the results show that after immobilization, OPA / UiO-66-NH2@LIP has a strong affinity for Zn 2+ , Ca 2+ and Cu 2+ The tolerance of Fe 2+ and Fe 3+ The OPA / UiO-66-NH2@LIP exhibited a strong inhibitory effect on free LIP, with the relative activity reduced by 70.29% and 69.16%, respectively. After immobilization, the OPA / UiO-66-NH2@LIP had a strong inhibitory effect on Fe 2+ and Fe 3+ The tolerance of OPA / UiO-66-NH2 was greatly improved, and the relative activity reached 97.13% and 125.46%, respectively. This indicates that OPA / UiO-66-NH2 immobilized enzyme can greatly reduce the negative effects of certain metal ions on enzyme activity and effectively improve the catalytic activity of the enzyme.

[0099] Organic solvent test: The free enzyme and immobilized enzyme were placed in 5 mL of methanol, ethanol, acetone, n-butanol, n-hexane, and dimethyl sulfoxide, respectively, and incubated at 40°C for 2 h. The changes in the activity of the free enzyme and the immobilized enzyme were observed. The activity test results are shown in 3e. The results show that OPA / UiO-66-NH2@LIP can maintain greater than 93% of its initial activity in the six organic solvents tested (methanol, ethanol, isopropanol, n-butanol, n-hexane, and DMSO), while free LIP almost loses its activity in ethanol and isopropanol, and only maintains about 30% of its initial activity in the remaining organic solvents.

[0100] High concentration surfactant test (i.e., mechanical stability): 0.8g of AEO9, FMEE, Brji, Tween-80, and Span-80 were dissolved in 10mL of PBS solution (0.1moL / L) to obtain 8wt% AEO9, 8wt% FMEE, 8wt% Brji, 8wt% Tween-80, and 8wt% Span-80 buffer solutions; the immobilized enzyme (OPA / UiO-66-NH2@LIP) was dispersed into the PBS solution and incubated on a shaker at 40°C for 2h, then washed twice with the above buffer solution. The effects of immobilization on enzyme stability before and after immobilization were investigated. The surfactant test results are shown in Figure 2. Figure 3 As shown in Figure 5, the results show that after mixing with a high concentration of surfactant and elution with anionic surfactant, the catalytic activity decreased significantly. The residual activity after 8% SDS was only about 30% of that before elution. However, the immobilized enzyme still showed good stability compared to the free enzyme. In contrast, the catalytic activity did not decrease significantly after elution with nonionic surfactant. In fact, 8% AEO9 increased the relative enzyme activity (114.29%). This shows that OPA / UiO-66-NH2@LIP is more stable than free LIP in all aspects.

[0101] Example 4

[0102] The difference from Example 1 is that the esterification reaction system does not contain a surfactant. In the biodiesel preparation process, the transesterification reaction time is set to 12 h, 24 h, 36 h, and 72 h, respectively; the transesterification reaction temperature is set to 30 degrees Celsius, 35 degrees Celsius, 45 degrees Celsius, and 50 degrees Celsius, respectively; the immobilized enzyme dosage is 30 mg, 60 mg, 120 mg, and 240 mg, respectively; the molar ratio of methanol to sunflower oil is 1:1, 2:1, 4:1, and 5:1, respectively. The biodiesel yield is as follows: Figure 4 As shown in the figure, the results show that when OPA / UiO-66-NH2@LIP is used as the catalyst, the optimal catalytic conditions for sunflower oil as the oil substrate are: catalytic temperature 40℃, catalytic time 48h, immobilized enzyme dosage 180mg, and methanol / sunflower oil molar ratio 3:1.

[0103] Example 5

[0104] The effect of surfactant on the catalytic efficiency of immobilized enzyme (OPA / UiO-66-NH2@LIP) on biodiesel was investigated. The difference from Example 1 was that 1.0 mL of 2% non-ionic surfactants of AEO9, FMEE, Brji, Span 80, and Tween 80 (0.2 g of AEO9, FMEE, Brji, Tween-80, and Span-80 were dissolved in 10 mL of PBS solution to obtain 2 wt% AEO9, 2 wt% FMEE, 2 wt% Brji, 2 wt% Tween-80, and 2 wt% Span-80 non-ionic surfactants, respectively) was added to the transesterification reaction system, and no non-ionic surfactant was added. The biodiesel yield was as follows: Figure 5 The results show that the addition of 1.0 mL of 2 wt% nonionic surfactant (FMEE) to the immobilized enzyme catalytic system merged the previously separated oil and organic phases. 2% FMEE effectively enhanced lipase activity. Furthermore, the addition of a low concentration of surfactant emulsified the immiscible oil substrate with methanol, reducing mass transfer limitations around the immobilized enzyme and thereby increasing the reaction rate.

[0105] Example 6

[0106] The reusability of free lipase, immobilized lipase, and surfactant-immobilized lipase (i.e., Free LIP, OPA / UiO-66-NH2@LIP, and OPA / UiO-66-NH2@LIP+2%FMEE) was investigated. Figure 6 As shown, the results show that as the number of free lipase uses increases, the yield of catalytic biodiesel synthesis gradually decreases (from 80.00% to 18.67%), and the catalytic activity is lost after the eighth cycle. In comparison, the immobilized enzyme exhibits good catalytic efficiency. After ten uses, the conversion rate of fatty acid methyl ester (FAME) can still reach 80.01%, and after thirteen uses, it can reach 49.21%. After adding a small amount of low-concentration non-ionic surfactant (FMEE) to the immobilized enzyme catalytic system, the catalytic efficiency is higher than that of OPA / UiO-66-NH2@LIP throughout the thirteen cycles, with the catalytic efficiency reaching a maximum of 99.15%, and reaching 84.12% after ten cycles.

[0107] Example 7

[0108] Other MOFs materials were used to immobilize lipase (LIP). 0.05 g of the MOFs materials shown in Table 1 (i.e., ZIF-8 (Zn), ZIF-67 (Co), MIL-53 (Al), MIL-88A (Fe), MIL-101 (Fe), PCN-224 (Cr), and UiO-66-NH2 (Cr)) were weighed and ultrasonically dispersed into 1 mL of isopropanol. 4 mL of a PBS mixed solution containing LIP was added. After being fixed on a shaker at a constant temperature, the mixture was centrifuged and the supernatant was taken out for later use. The product after centrifugation was washed with PBS until there was no protein residue in the supernatant, and then freeze-dried for 12 h to obtain the immobilized enzyme. The relative enzyme activity and enzyme loading of the immobilized enzyme are shown in Table 1. The results show that the UiO-66-NH2 immobilized lipase has a high affinity that is significantly better than that of the other MOFs, and the modified method of the present invention can significantly improve the catalytic activity and enzyme loading of fat rice.

[0109] Table 1 Enzyme activity and enzyme loading of lipase after immobilization on different MOF materials

[0110]

[0111]

[0112] The above descriptions are merely several embodiments of the present invention and do not constitute any form of limitation to the present invention. Although the present invention is disclosed as above in terms of preferred embodiments, they are not intended to limit the present invention. Any technician familiar with the present profession who, without departing from the scope of the technical solution of the present invention, makes slight changes or modifications using the technical contents disclosed above are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. An immobilized lipase for preparing biodiesel, characterized in that: The immobilized lipase comprises a metal organic framework carrier modified with a hydrophobic group and a lipase immobilized on the metal organic framework carrier; The lipase immobilized on the metal organic framework support is used in combination with a nonionic surfactant as a co-catalyst; The hydrophobic group is selected from OPA.

2. The immobilized lipase according to claim 1, characterized in that The metal organic framework is selected from at least one of the ZIF series, MIL series, PCN series, and UiO series; Preferably, the metal organic framework is of the UiO series; More preferably, the metal organic framework is UiO-66-NH2.

3. The immobilized lipase according to claim 1, characterized in that The metal organic framework is UiO-66-NH2, and the hydrophobic group is OPA, wherein the mass ratio of UiO-66-NH2 to OPA is 50:1-5, and OPA is connected to the surface of UiO-66-NH2 through a covalent bond to obtain a metal organic framework carrier.

4. The immobilized lipase according to claim 1, characterized in that The mass ratio of the lipase to the metal organic framework carrier is 1-5:5-10; The nonionic surfactant is selected from at least one of fatty acid methyl ester ethoxylate, fatty alcohol polyoxyethylene ether, polyoxyethylene lauryl alcohol, Span-80, and Tween-80; Preferably, the nonionic surfactant is fatty acid methyl ester ethoxylate; The mass concentration of the nonionic surfactant is 0.5-2%; The mass ratio of the nonionic surfactant to the immobilized enzyme is 1-4:6-48.

5. A method for preparing the immobilized lipase according to any one of claims 1 to 4, characterized in that: The preparation method comprises at least the following steps: Step 1, synthesis of UiO-66-NH2: zirconium chloride and 2-aminoterephthalic acid are dissolved in N,N-dimethylformamide, ultrasonically dissolved, acetic acid and water are added, stirred, then N,N-dimethylformamide is added, stirred, and transferred to a hydrothermal reactor for reaction to obtain an orange-red suspension. The product is collected by centrifugation, washed with N,N-dimethylformamide and methanol, and vacuum dried to obtain UiO-66-NH2; Step 2, synthesis of OPA / UiO-66-NH2: dissolve OPA in ethanol, disperse by ultrasonication, add UiO-66-NH2 obtained in step 1, react at room temperature for 24 to 48 hours, stirring for 5 minutes every 2 hours, wash the reaction product by centrifugation with ethanol, and dry in vacuo to obtain OPA / UiO-66-NH2; Step 3, immobilization of lipase: ultrasonically disperse the OPA / UiO-66-NH2 obtained in step 2 into isopropanol, add PBS solution containing lipase, shake well and place on a shaker, react, centrifuge, wash, and freeze-dry at degrees Celsius to obtain immobilized lipase.

6. The preparation method according to claim 5, characterized in that In step 2, the mass mixing ratio of OPA and UiO-66-NH2 is 50:1-5; The reaction conditions in step 3 are as follows: The reaction time is 0.5 to 1 h; The reaction temperature is 30 to 50 degrees Celsius; The reaction was carried out at a speed of 150 to 180 rpm; The concentration of the lipase in the PBS solution containing lipase in step 3 is 2-5 mg / mL.

7. Use of the immobilized lipase according to any one of claims 1 to 4 and / or the immobilized lipase prepared by the preparation method according to any one of claims 5 to 6 in the preparation of biodiesel, characterized in that: The immobilized lipase is mixed with the oil substrate, a co-solvent is added, preheated, and an organic alcohol is added to carry out an ester exchange reaction. After the reaction is completed, the immobilized lipase is separated by centrifugation and the biodiesel is evaporated.

8. The use according to claim 7, characterized in that The oil substrate is selected from at least one of sunflower oil, rapeseed oil, and olive oil; The co-solvent is selected from at least one of n-hexane, toluene, and tetrahydrofuran; The mass mixing ratio of the immobilized enzyme to the oil substrate is 15-120:

264.

9. The use according to claim 7, characterized in that The preheating temperature is 35 to 45 degrees Celsius; The organic alcohol is selected from at least one of methanol, ethanol, propanol and butanol; The volume ratio of the organic alcohol to the oil substrate is 1 to 5:1; The conditions for the transesterification reaction of the organic alcohol and the ester exchange reaction are as follows: The transesterification reaction time is 12 to 72 hours; The temperature of the transesterification reaction is 30 to 50 degrees Celsius.

10. The use according to claim 7, characterized in that Adding a nonionic surfactant before the transesterification reaction; The nonionic surfactant is selected from at least one of fatty acid methyl ester ethoxylate, fatty alcohol polyoxyethylene ether, polyoxyethylene lauryl alcohol, Span-80, and Tween-80; The mass concentration of the nonionic surfactant is 0.5-2%; The mass ratio of the nonionic surfactant to the immobilized lipase is 1-4:6-48.

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