Method for preparing immobilized candida antarctica lipase

Abstract

The application belongs to the technical field of immobilized enzyme, and particularly relates to a method for preparing immobilized Candida antarctica lipase. The preparation method is as follows: 2-methyl imidazole is dissolved in deionized water, and Candida antarctica lipase solution is added and uniformly mixed; zinc acetate dihydrate is dissolved in deionized water; then the zinc acetate dihydrate solution is quickly added into the enzyme-containing solution, and after oscillation and mixing, the solution is placed at room temperature for reaction; then the precipitate is collected by centrifugation, and is washed by deionized water and anhydrous ethanol respectively, and finally, the precipitate is dried at room temperature under reduced pressure to obtain CALB@ZIF-8 immobilized enzyme. The immobilized Candida antarctica lipase prepared by the method has excellent organic solvent resistance, storage stability and reusability.

Description

Technical Field

[0001] This invention belongs to the technical field of immobilized enzymes, specifically relating to a method for preparing immobilized Candida antarcticis lipase. Background Technology

[0002] Antarctic Candida lipase B (CALB) possesses advantages such as high catalytic activity, broad substrate applicability, and good stereoselectivity, showing great promise for applications in oil modification, biodiesel production, ester synthesis, and pharmaceutical intermediate preparation. However, free lipases suffer from poor stability, insufficient heat and organic solvent resistance, and difficulty in recovery and reuse, limiting their industrial application. Therefore, immobilization of lipases has become an important technical approach to improve their stability and reduce production costs.

[0003] Currently, lipase immobilization supports mainly include natural polymers, inorganic porous materials, organic polymers, and metal-organic frameworks (MOFs). Among them, traditional supports such as sodium alginate, chitosan, silica gel, and resins have advantages such as mature preparation processes and low costs, but they generally suffer from problems such as limited enzyme loading capacity, easy leakage of enzyme molecules, low mass transfer efficiency, insufficient mechanical stability, and rapid decline in enzyme activity during repeated use. When using covalent immobilization, the cross-linking agent may also react with the enzyme active site, leading to partial loss of enzyme activity.

[0004] In recent years, metal-organic frameworks (MOFs) have been widely used in enzyme immobilization due to their advantages such as large specific surface area, high porosity, tunable pore size, designable composition, and ease of surface functionalization. Among them, zeolite imidazolium ester framework material-8 (ZIF-8) can achieve in-situ encapsulation of enzyme molecules through biomimetic mineralization, effectively improving enzyme loading, catalytic activity, and stability. Previous studies have shown that MOFs such as ZIF-8, UiO-66, MIL-88A, and Ce-MOF can be used to immobilize lipases from different sources, and have improved catalytic efficiency and cycling stability in some systems.

[0005] Unlike traditional adsorption, covalent bonding, and encapsulation methods, this invention employs a biomimetic mineralization in-situ encapsulation strategy, enabling the simultaneous formation of MOF materials and Candida antarcticis lipase. Specifically, during the nucleation and growth of MOF crystals, the lipase is encapsulated within the MOF framework as a biological template, rather than preparing MOFs first and then performing enzyme adsorption and immobilization. This method avoids problems in traditional immobilization processes, such as the difficulty of large enzyme molecules entering the carrier due to pore size limitations, enzyme molecule detachment due to weak adsorption, and damage to enzyme active sites due to chemical cross-linking during covalent immobilization. It also effectively reduces conformational changes of the enzyme during immobilization, improving encapsulation efficiency and activity retention. The biomimetic mineralization-formed MOF shell also provides a stable protective layer for the enzyme molecules, allowing them to maintain high catalytic activity under conditions such as high temperature, organic solvents, and long-term storage. Its protective effect is significantly superior to traditional inorganic supports such as CaCO3 and porous silica.

[0006] However, MOFs are not suitable for all lipase systems. Due to differences in molecular size, surface charge, hydrophobicity, and conformational characteristics among different lipases, the immobilization effect of the same MOF material varies significantly for different lipases. Even when using the same MOF carrier, immobilized enzymes obtained through different immobilization methods and surface modification strategies still exhibit considerable differences in catalytic activity, stability, and recyclability. Currently, research on MOF immobilization of Candida antarcticis lipase still faces challenges such as limited immobilization efficiency, insufficient regulation of the enzyme microenvironment during immobilization, and the need for further improvement in cycling stability. In particular, there is a lack of research on improving enzyme immobilization performance by regulating the nucleation and growth process of MOF crystals.

[0007] Therefore, developing an immobilization method that can regulate the crystal formation process of MOFs, improve the microenvironment of immobilized enzymes, and further enhance the catalytic performance and cycling stability of Candida antarcticis lipase is of great research significance and application value. Summary of the Invention

[0008] To address the aforementioned technical issues, this invention utilizes metal-organic frameworks (MOFs) to prepare immobilized Candida antarcticis lipase. The results show that the immobilized Candida antarcticis lipase exhibits excellent storage stability and reusability. A recycling experiment was conducted in this invention, demonstrating that the enzyme activity remained above 75% after 10 reuses, significantly improving the cyclic stability of the immobilized lipase.

[0009] The immobilized Candida antarcticis lipase prepared by the method of this invention exhibits high enzyme activity retention and significantly improved resistance to organic solvents, thermal stability, storage stability, and reusability. Specifically, it retains approximately 80%–90% relative enzyme activity in polar organic solvents such as methanol, ethanol, and isopropanol; after 60 days of storage at room temperature, it retains approximately 69% relative enzyme activity; and after 10 consecutive cycles, the relative enzyme activity remains above 75%, significantly superior to the undoped system, demonstrating promising prospects for industrial application.

[0010] The method for preparing immobilized Candida antarcticis lipase described in this invention includes the following steps:

[0011] (1) Mix 2-methylimidazole with deionized water to obtain a 2-methylimidazole solution;

[0012] (2) Add Candida antarctica lipase solution to the 2-methylimidazole solution in (1) and mix well. This solution is called solution I.

[0013] (3) Dissolve zinc acetate dihydrate in deionized water to obtain zinc acetate dihydrate solution;

[0014] (4) Add the zinc acetate dihydrate solution from (3) to solution I, shake to mix, and then let it stand at room temperature to react;

[0015] (5) After the reaction was completed, the precipitate was collected by centrifugation, washed with deionized water and anhydrous ethanol respectively, and dried under reduced pressure at room temperature to obtain CALB@ZIF-8 immobilized enzyme.

[0016] Preferably, (1) the mass-to-volume ratio of 2-methylimidazole to deionized water is (0.40-0.50) g: (12-18) mL.

[0017] Preferably, in (2), the concentration of Candida antarcticis lipase solution is 1-2 mg / mL; the volume ratio of Candida antarcticis lipase solution to 2-methylimidazole solution is (12-18):(4-6) mL.

[0018] Preferably, in (3), the mass-to-volume ratio of zinc acetate dihydrate to deionized water is (0.16-0.20) g:(12-18) mL.

[0019] Preferably, in (4), after shaking and mixing for 8-12s, the mixture is allowed to stand at room temperature for 10-14h; the volume ratio of zinc acetate dihydrate solution to solution I is (12~18) mL: (16~24) mL.

[0020] Preferably, the centrifugation speed is 5000-7000 rpm; the centrifugation time is 8-12 min; and the centrifugation is performed twice with deionized water and anhydrous ethanol, respectively.

[0021] Preferably, L-histidine is added to the 2-methylimidazole solution in (1) in an amount of 10-30% of the mass of the 2-methylimidazole solution, and then (2) is carried out.

[0022] Preferably, the method for preparing immobilized Candida antarcticis lipase is characterized by comprising the following steps:

[0023] (1) Mix 2-methylimidazole with deionized water at a mass-to-volume ratio of (0.40-0.50) g:(12-18) mL to obtain a 2-methylimidazole solution;

[0024] (2) Add 1-2 mg / mL Candida antarcticis lipase solution to the 2-methylimidazole solution in (1) and mix well; the volume ratio of Candida antarcticis lipase solution to 2-methylimidazole solution is (12~18):(4~6) mL; denoted as solution I;

[0025] (3) Dissolve zinc acetate dihydrate in deionized water to obtain zinc acetate dihydrate solution; the mass-to-volume ratio of zinc acetate dihydrate to deionized water is (0.16-0.20) g:(12-18) mL;

[0026] (4) Add the zinc acetate dihydrate solution from (3) to solution I, shake and mix for 8-12 seconds, and then let it stand at room temperature for 10-14 hours. The volume ratio of zinc acetate dihydrate solution to solution I is (12~18) mL: (16~24) mL.

[0027] (5) After the reaction was completed, the precipitate was collected by centrifugation at 5000-7000 rpm for 8-12 min, washed twice with deionized water and anhydrous ethanol, and finally dried under reduced pressure at room temperature to obtain CALB@ZIF-8 immobilized enzyme.

[0028] Preferably, the method for preparing immobilized Candida antarcticis lipase includes the following steps:

[0029] (1) Mix 2-methylimidazole with deionized water at a mass-to-volume ratio of (0.40-0.50) g:(12-18) mL to obtain a 2-methylimidazole solution; then add L-histidine in an amount of 10-30% of the mass of the 2-methylimidazole solution; this is referred to as solution II.

[0030] (2) Add 1-2 mg / mL Candida antarcticis lipase solution to the solution obtained in (1) and mix well; the volume ratio of Candida antarcticis lipase solution to 2-methylimidazole solution is (12~18):(4~6) mL; denoted as solution I;

[0031] (3) Dissolve zinc acetate dihydrate in deionized water to obtain zinc acetate dihydrate solution; the mass-to-volume ratio of zinc acetate dihydrate to deionized water is (0.16-0.20) g:(12-18) mL;

[0032] (4) Add the zinc acetate dihydrate solution from (3) to solution I, shake and mix for 8-12 seconds, and then let it stand at room temperature for 10-14 hours. The volume ratio of zinc acetate dihydrate solution to solution I is (12~18) mL: (16~24) mL.

[0033] (5) After the reaction was completed, the precipitate was collected by centrifugation at 5000-7000 rpm for 8-12 min, washed twice with deionized water and anhydrous ethanol, and finally dried under reduced pressure at room temperature to obtain CALB@ZIF-8 immobilized enzyme.

[0034] In this invention, MOFs materials are actually formed at the same time as the immobilized lipase (CALB). The specific principle is that the MOFs are constructed in situ using zinc salt and 2-methylimidazole through a biomimetic mineralization method to form ZIF-8, and Antarctic Candida lipase (CALB) is simultaneously encapsulated during the formation of MOFs to achieve enzyme immobilization.

[0035] As a preferred approach, in the biomimetic mineralization process, L-histidine is added to a solution of 2-methylimidazolium organic ligands, followed by the addition of *Candida antarcticis* lipase solution, and then mixed with a zinc salt solution for in-situ mineralization. The imidazole and amino groups in L-histidine can react with Zn... 2+ Coordination occurs, regulating Zn during the nucleation and growth of MOF crystals. 2+ The local coordination environment of the enzyme can compete with or cooperate with 2-methylimidazole to regulate the nucleation rate and growth behavior of the crystal, improve the crystal structure, particle size distribution and pore structure of MOFs, and build a microenvironment that is more conducive to the enzyme molecules maintaining their native conformation during the immobilization process. This reduces the conformational changes of the enzyme molecules during the mineralization process and improves the enzyme's encapsulation efficiency, substrate accessibility, catalytic activity, thermal stability and reusability.

[0036] The beneficial effects of this invention are as follows:

[0037] (1) The immobilized Candida antarcticis lipase prepared by the method of the present invention has excellent organic solvent tolerance, storage stability and reusability; specifically, in polar solvents such as methanol, ethanol and isopropanol, the immobilized enzyme can still maintain about 80-90% of its activity; after 60 days, it still retains about 69% of the relative enzyme activity; after 10 reuses, the enzyme activity is still higher than 75%;

[0038] (2) The present invention adopts a biomimetic mineralization in-situ embedding strategy, which has a simple preparation process, mild conditions, no need for complex chemical cross-linking process, can effectively reduce enzyme activity loss, and has good repeatability and scale-up application potential.

[0039] (3) This invention introduces L-histidine into the ZIF-8 biomimetic mineralized immobilized Candida antarctic lipase system for the first time. By regulating the nucleation, growth and enzyme microenvironment of MOFs crystals, the synergistic improvement of the catalytic performance and cycle stability of the immobilized enzyme is achieved, providing a new technical solution for the design of MOFs immobilized lipase carriers. Attached Figure Description

[0040] Figure 1 The left image shows the p-NP standard curve; the right image shows the quantitative standard curve for proteins.

[0041] Figure 2 The results of CALB immobilization condition optimization;

[0042] (A) shows the effect of enzyme concentration on the carrier encapsulation efficiency; (B) shows the effect of L-HIS doping amount on the carrier encapsulation efficiency.

[0043] (C) shows the effect of immobilization time on the carrier encapsulation efficiency; (D) shows the effect of buffer pH on the carrier encapsulation efficiency.

[0044] Figure 3 Scanning electron microscope images of CALB@ZIF-8 and CALB@L-His@ZIF-8;

[0045] Figure 4 XRD plots for CALB@ZIF-8 and CALB@L-His@ZIF-8;

[0046] Figure 5 FTIR spectra of CALB, CALB@ZIF-8, and CALB@L-HIS@ZIF-8;

[0047] Figure 6 The N2 adsorption-desorption isotherms for CALB@ZIF-8 and CALB@L-His@ZIF-8 are shown.

[0048] Figure 7 Thermogravimetric analysis results of free and immobilized CALB enzymes;

[0049] Figure 8 This is a zeta potential diagram of CALB lipase;

[0050] Figure 9 For enzyme property analysis;

[0051] (A) Optimal temperatures of free enzymes and immobilized lipases; (B) Temperature stability of free enzymes and immobilized lipases;

[0052] (C) Optimal pH of free enzymes and immobilized lipases; (D) pH stability of free enzymes and immobilized lipases;

[0053] Figure 10 To enhance the organic solvent tolerance of both free enzymes and immobilized lipases;

[0054] Figure 11 For protein secondary structure analysis;

[0055] Figure 12 To improve the storage stability of immobilized lipase;

[0056] Figure 13 To ensure the reusability of immobilized lipase. Detailed Implementation

[0057] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0058] 1. Materials and reagents of this invention: Candida antarcticis lipase B (Novozymes (China) Biotechnology Co., Ltd.); zinc acetate dihydrate (Guangdong Saiketong Technology Co., Ltd.); 2-methylimidazole (Shanghai Maclean Biotechnology Co., Ltd.); L-histidine (Shanghai Titan Technology Co., Ltd.); BCA protein content reagent kit (Beijing Box Biotechnology Co., Ltd.); p-nitrophenol (Shanghai E-En Chemical Technology Co., Ltd.); p-nitrophenol palmitate (Shanghai Maclean Biotechnology Co., Ltd.).

[0059] 2. Instruments and Equipment: Instruments and Equipment

[0060] AY220 analytical balance (Mettler-Toledo, Switzerland); SHA-B water bath (Shanghai Jingke Co., Ltd.); SB25-12DT ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); TG16 high-speed centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.); Synergry H1 microplate reader (BioTek, USA); SCIENTZ-12N / C vacuum freeze dryer (Ningbo Xinzhi Biotechnology Co., Ltd.); Nicolet-iS5 Fourier transform infrared spectrometer (Thermo Fisher Scientific, USA); Regulus 8100 scanning electron microscope (Hitachi, Inc.); ASAP2460 surface area and porosity analyzer (McMerrittek (Shanghai) Instrument Co., Ltd.); STA449F5 thermogravimetric analyzer (NETZSCH Instruments Manufacturing Co., Ltd.)

[0061] Unless otherwise specified, all other materials or instruments are commercially available.

[0062] Example 1

[0063] 1. Preparation of biomimetic mineralized CALB lipase

[0064] (1) Preparation of biomimetic mineralized CALB@ZIF-8 lipase

[0065] Immobilized lipase CALB@ZIF-8 was prepared using a biomimetic mineralization method. 0.436 g of 2-methylimidazole was dissolved in 15 mL of deionized water, and a certain amount of CALB enzyme solution was added and mixed thoroughly. Separately, 0.176 g of zinc acetate dihydrate was dissolved in 15 mL of deionized water. The zinc source solution was then rapidly added to the enzyme-containing ligand solution, and the mixture was shaken for 10 s and allowed to stand at room temperature for 12 h. After the reaction was complete, the precipitate was collected by centrifugation at 6000 rpm for 10 min, washed twice with deionized water and anhydrous ethanol, and finally dried under reduced pressure at room temperature to obtain the CALB@ZIF-8 immobilized enzyme.

[0066] (2) Preparation of biomimetic mineralized CALB@L-HIS@ZIF-8 lipase

[0067] The preparation method of CALB@L-HIS@ZIF-8 is basically the same as that of CALB@ZIF-8. The difference is that L-HIS is added to the ligand solution in different molar ratios of 2-methylimidazole, and after being fully dissolved with 2-methylimidazole, CALB enzyme solution is added. The remaining reaction and post-processing steps are the same as the above method, and finally CALB@L-HIS@ZIF-8 immobilized enzymes with different L-HIS doping amounts are obtained.

[0068] 2. Optimization of biomimetic mineralized CALB lipase immobilization conditions

[0069] The key factors in the enzyme immobilization process (enzyme concentration, L-HIS doping amount, immobilization time, and buffer pH) in point 1 were optimized. Specific conditions were as follows: enzyme concentration (0.25, 0.5, 1.25, 2.0, 5.0 mg / mL); L-HIS doping amount (0%, 10%, 20%, 30%, 40%, 50%); immobilization time (2, 6, 12, 18, 24 h); buffer pH (6.0, 7.0, 8.0, 9.0, 10.0), the same as in point 1. Finally, the protein concentration before and after immobilization under each single-factor condition was measured, and the enzyme loading rate was calculated.

[0070] 3. Lipase activity assay

[0071] (1) Preparation of p-nitrophenol (p-NP) standard curve

[0072] Prepare a 0.1 mg / mL p-NP standard solution. Take 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mL of the p-NP standard solution into test tubes, and dilute to 10 mL with Tris-HCl buffer at pH 7.0. Measure the absorbance OD404. Plot a standard curve with OD404 as the x-axis and the amount of p-NP as the x-axis.

[0073] (2) Lipid hydrolase activity assay system

[0074] Lipid hydrolase (esterase / lipase) activity was determined using the p-NP hydrolysis colorimetric method. The p-nitrophenol produced by the hydrolysis of the substrate by lipohydrolases is yellow in water and has a maximum absorbance at 404 nm; therefore, the enzyme activity of lipohydrolases can be calculated by measuring the amount of this product produced. The specific determination method is as follows: Substrate solution: Prepare a 10 mmol p-nitrophenyl ester (p-NPCester) substrate stock solution using acetonitrile solution and store at 4°C for later use.

[0075] Enzyme activity assay: Add 20 mL of 10 mmol / L substrate solution to 880 L of standard phosphate buffer or 50 mmol / L Tris-HCl buffer with a specific pH, incubate at 37°C for 5–10 min, then add 100 μL of appropriately diluted enzyme solution, bringing the total reaction volume to 1 mL. React for 10 min, and immediately measure the absorbance at OD404. Use an enzyme solution inactivated under the same conditions as a blank control. Calculate enzyme activity based on the p-nitrophenol standard curve. Enzyme activity unit definition: Under certain conditions, the amount of enzyme required per minute for a lipase (esterase / lipase) to decompose a substrate and produce 1 μmol of p-nitrophenol is defined as one enzyme activity unit (U). Enzyme activity calculation formula:

[0076]

[0077] In the formula:

[0078] (axΔOD+b): The y-value of the p-nitrophenol standard curve plotted with OD404 as the X-axis and the amount of p-NP as the Y-axis.

[0079] F: Dilution factor of enzyme solution:

[0080] 10: Coefficient converted to 1 mL of reaction system

[0081] T: Reaction time (min);

[0082] 0.1: Enzyme solution volume.

[0083] 4. Lipase protein concentration determination

[0084] The encapsulation efficiency of the enzyme protein in the immobilized system was determined using the BCA method. Samples were appropriately diluted with PBS buffer before testing. A BCA working solution was prepared at a BCA Solution:Copper Solution ratio of 50:1 (V / V), and thoroughly mixed before use. 10 μL of BSA standard solution (5 mg / mL) was diluted to 100 μL with PBS to obtain a 0.5 mg / mL protein standard dilution, which was further diluted to a series of standard solutions of 0, 0.05, 0.10, 0.15, 0.20, 0.30, 0.40, and 0.50 mg / mL. 20 μL of the diluted sample or different concentrations of standard solution were added to each well of a 96-well plate, followed by 200 μL of BCA working solution. The mixture was incubated at 37℃ for 15–30 min. The absorbance at 562 nm was measured using the corresponding standard solution as a reference. A standard curve was plotted with the standard solution concentration as the abscissa (x, mg / mL) and absorbance as the ordinate (y), yielding the linear regression equation y = kx + b. The protein concentration was calculated by substituting the sample absorbance into the equation, and the enzyme protein content in the stock solution was converted according to the dilution factor, thus calculating the encapsulation efficiency.

[0085] 5. Characterization of immobilized lipase

[0086] (1) Scanning electron microscopy analysis

[0087] The morphology of the samples was observed using a field emission scanning electron microscope (SEM) of model SU8010 (Hitachi, Japan), with an accelerating voltage of 5 kV. Before testing, the samples were sputtered with gold, then ultrasonically dispersed in deionized water for 5 min, dropped onto the surface of mica sheets, and allowed to air dry before testing.

[0088] (2) X-ray diffraction analysis

[0089] X-ray diffraction (XRD) was used to analyze the crystal structure and evolution of the samples. An EMPYREAN X-ray diffractometer (Cu target, Ni filter) was used, with Cu Kα rays as the radiation source at a wavelength of λ = 0.15418 nm. The test conditions were: tube voltage 40 kV, tube current 40 mA, scan range 2θ = 5°–60°, step size 0.02°, and scan rate 2° / min.

[0090] (3) Fourier transform infrared analysis

[0091] The functional groups of CALB, CALB@ZIF-8, and CALB@L-HIS@ZIF-8 were characterized using FTIR to determine the preparation of the support and the L-HIS modification. FTIR measurements were performed on a Nicolet iS50 infrared spectrometer using the KBr pellet method, with a wavenumber range of 400–4000 cm⁻¹. -1Characteristic peaks of CALB, CALB@ZIF-8, and CALB@L-HIS@ZIF-8 were detected.

[0092] (4) N2 adsorption-desorption analysis

[0093] The specific surface area and pore structure of CALB@ZIF-8 and CALB@L-HIS@ZIF-8 were analyzed using the BET method to examine the changes in surface area and pore structure before and after L-HIS modification. Nitrogen adsorption-desorption data were obtained using a Micron ASAP2460 surface area and pore size analyzer (USA), and the tests included specific surface area, mesopore and micropore analysis.

[0094] (5) Thermogravimetric analysis

[0095] Thermogravimetric analysis (TGA) was used to analyze the thermal stability and compositional changes of the samples. The tests were conducted using a thermogravimetric analyzer under a nitrogen atmosphere, with approximately 10 mg of sample as the test object. The heating rate was 10 °C / min, and the test temperature range was 30–800 °C. The thermal stability and organic component content of the material were analyzed by recording the changes in sample mass with temperature.

[0096] (6) Immobilized lipase Zeta potential

[0097] The adsorbent material was dissolved in anhydrous ethanol and sonicated for at least 5 minutes. Then it was added to a cuvette and the zeta potential of the sample was detected using a 90PLUS (USA) instrument.

[0098] 6. Enzymatic property analysis

[0099] (1) Optimal temperature and temperature stability of immobilized lipase

[0100] The thermostability of immobilized lipases was evaluated using a residual enzyme activity assay after heat treatment, and compared with that of free enzymes. Free CALB, CALB@ZIF-8, and CALB@L-His@ZIF-8 enzymes with the same initial enzyme activity were dispersed in PBS buffer (25 mM, pH 7.0) and incubated at 40, 50, 60, 70, and 80 °C for 2 h, respectively; simultaneously, they were incubated at 60 °C for 0, 2, 4, 6, 8, and 10 h to investigate their long-term thermostability. After treatment, enzyme activity was measured at their respective optimal reaction temperature and pH, and the activity before heat treatment was defined as 100%. Relative enzyme activity was calculated to assess the effects of immobilization and L-His modification on the thermostability of lipases.

[0101] (2) Optimal pH and pH stability of immobilized lipase

[0102] The pH stability of immobilized lipases was evaluated using residual enzyme activity assays under different pH conditions, and compared with that of free enzymes. First, PBS buffers with pH ranges of 4-12 were prepared, and the activities of free CALB, CALB@ZIF-8, and CALB@L-His@ZIF-8 were measured under specific temperature conditions to screen for their optimal reaction pH. Subsequently, enzyme samples with the same initial activity were dispersed in PBS buffers at different pH ranges (4-12) and incubated at 25°C for 2 hours. Long-term incubation experiments at 0, 2, 4, 6, 8, and 10 hours were also conducted to examine changes in pH stability. After treatment, enzyme activity was measured at their respective optimal reaction temperatures and pH conditions. The highest enzyme activity was defined as 100% relative activity, and residual activity was calculated to evaluate the effects of immobilization and L-His modification on the pH stability of lipases.

[0103] (3) Organic solvent tolerance of immobilized lipase

[0104] The organic solvent tolerance of immobilized lipases was evaluated using a residual enzyme activity assay after treatment with different organic solvents. Free CALB, CALB@ZIF-8, and CALB@L-His@ZIF-8 lipases with the same initial enzyme activity were dispersed in PBS buffer and different organic solvents (methanol, ethanol, tert-amyl alcohol, isopropanol, n-hexane, ethyl acetate, and acetonitrile), respectively, and incubated at 60°C for 2 h. After treatment, the hydrolytic activity of each sample was measured according to the enzyme activity assay method. The enzyme activity in PBS without organic solvent treatment was defined as 100% relative activity, and the residual activity of each system was calculated to evaluate the effect of immobilization and L-His modification on the organic solvent stability of lipases.

[0105] (4) Storage stability of immobilized lipase

[0106] The storage stability of immobilized lipases was evaluated using a residual enzyme activity assay after long-term low-temperature storage, and compared with that of free enzymes. CALB@ZIF-8 and CALB@L-His@ZIF-8 were stored at 4°C, and their hydrolytic enzyme activities were measured at 0, 5, 10, 15, 20, 25, 30, and 60 days. Enzyme activity assays were performed at their respective optimal reaction temperatures and pH values, and the initial enzyme activity was defined as 100% relative activity. Residual activity was calculated at different storage times to assess the effects of immobilization and L-His modification on the storage stability of the lipases.

[0107] (5) Reusability of immobilized lipase

[0108] Immobilized enzymes prepared using ZIF-8 and L-His@ZIF-8 as carriers exhibit good structural stability and recyclability, are easily separated from the reaction system, and have the potential for multiple recycling. Therefore, their reusability was evaluated. Since free CALB is difficult to recover from the reaction system and its activity is easily affected by the operational process, the reusability of the free enzyme was not tested. Under optimal immobilization and reaction conditions, CALB@ZIF-8 and CALB@L-His@ZIF-8 were subjected to a 10-cycle reuse experiment. After each reaction, the immobilized enzyme was centrifuged, washed with n-hexane, and then added back to a fresh reaction system for the next cycle. The enzyme activity measured in the first cycle was defined as 100% relative activity, and the residual activity in subsequent cycles was calculated to evaluate the reusability of the immobilized enzyme.

[0109] Example 2

[0110] 2.1 Optimization of CALB Immobilization Conditions

[0111] 2.1.1 Effect of enzyme concentration on vector encapsulation efficiency

[0112] When the enzyme concentration reached approximately 1.25–2.5 mg / mL, the encapsulation efficiency of ZIF-8 reached its peak (75%), while that of L-HIS@ZIF-8 was slightly lower (70%). This indicates that L-HIS doping slightly reduced the crystallization rate and encapsulation density of ZIF-8, but its subsequent enzyme activity retention and stability may be better.

[0113] 2.1.2 Effect of L-HIS doping concentration on carrier encapsulation efficiency

[0114] The encapsulation efficiency of each system remained within the range of approximately 60%–75%, with relatively small fluctuations. As the L-His doping ratio increased from 10% to 30%, the relative enzyme activity of the immobilized enzymes showed a significant upward trend, with the 30% L-His@ZIF-8 system achieving the highest encapsulation efficiency, significantly higher than other doping ratios.

[0115] 2.1.3 Effect of immobilization time on carrier encapsulation efficiency

[0116] The encapsulation efficiency of the ZIF-8 and L-HIS@ZIF-8 systems gradually increased from 2 to 12 hours, reaching a maximum at 12 hours, and then decreased slightly with the extension of time, eventually stabilizing.

[0117] 2.1.4 Effect of buffer pH on carrier encapsulation efficiency

[0118] Encapsulation efficiency increased with increasing pH in both systems within the pH range of 6–8, reaching its maximum at pH 8; excessively high pH was detrimental to framework stability. Overall, pH 8 was the optimal biomineralization condition, achieving high enzyme encapsulation efficiency while ensuring stable framework construction.

[0119] 2.2 Characterization of immobilized lipase

[0120] 2.2.1 Scanning electron microscopy analysis

[0121] SEM results show that CALB@ZIF-8 exhibits relatively uniform nanoparticles and partially retains a polyhedral structure, demonstrating typical ZIF-8 biomimetic mineralization characteristics. In contrast, CALB@L-His@ZIF-8 exhibits aggregates without obvious crystal faces and with disordered structure, indicating that L-His alters the nucleation and growth path of ZIF-8 and promotes the formation of amorphous structures.

[0122] 2.2.2 X-ray diffraction analysis

[0123] XRD results showed that neither CALB@ZIF-8 nor CALB@L-His@ZIF-8 exhibited the characteristic diffraction peaks of typical ZIF-8, but instead displayed obvious broad peaks, indicating that the materials have low crystallinity and belong to an amorphous or low-crystalline structure. Especially after the introduction of L-His, the ZIF-8 crystal structure was further weakened, possibly due to the interaction between L-His and Zn. 2+ The competitive coordination between them inhibits the orderly assembly of the Zn-2-MIM framework.

[0124] 2.2.3 Fourier Transform Infrared Analysis

[0125] CALB at 3200–3500cm -1 A distinct –OH stretching vibration peak appears at approximately 2920 cm⁻¹. -1 A C–H stretching vibration peak appears at approximately 1650 cm⁻¹, while at approximately 1650 cm⁻¹... -1 The characteristic absorption peak of NH (amide I band) appears at this location, which is a typical feature of protein structure. Compared with free CALB, CALB@ZIF-8 and CALB@L-HIS@ZIF-8 still retain these characteristic peaks at the corresponding positions, indicating that the basic structure of the enzyme was not significantly destroyed during immobilization. Meanwhile, the characteristic peak of the imidazole ligand (1580 cm⁻¹) is also present. -1 1140-1300cm -1 ) and Zn-N vibration peak (420-450 cm) -1 The presence of ) indicates the existence of a Zn-imidazolium coordination structure in the system. Compared to CALB@ZIF-8, CALB@L-HIS@ZIF-8 exhibits better performance at 3200–3400 cm⁻¹. -1and 1600cm -1 The slight enhancement of the nearby absorption peaks suggests that the introduction of L-histidine may interact with the enzyme and the ZIF-8 backbone through hydrogen bonding or coordination, thereby promoting the stable immobilization of the enzyme.

[0126] 2.2.4 N2 Adsorption-Desorption Analysis

[0127] Both CALB@ZIF-8 and CALB@L-His@ZIF-8 exhibited rapid adsorption in the low relative pressure region (P / P0 < 0.1), indicating that the materials retain a certain degree of microporous structure. With increasing relative pressure, an increase in adsorption and a slight hysteresis loop appeared in the high-pressure region (P / P0 ≈ 0.9-1.0), indicating the presence of a certain degree of mesoporous structure in the system, mainly originating from particle packing or structural defects. In contrast, CALB@L-His@ZIF-8 showed a slightly higher adsorption capacity, suggesting that the introduction of L-His modulates the pore structure to some extent, making it more open and beneficial for substrate diffusion and catalytic reactions.

[0128] 2.2.5 Thermogravimetric Analysis

[0129] As shown in the figure, free CALB undergoes significant weight loss in the temperature range of approximately 200-320°C, mainly attributed to the thermal decomposition of the protein molecular structure. In contrast, the biomimetic mineralized samples CALB@ZIF-8 and CALB@L-His@ZIF-8 exhibit a more stable mass change trend over a wider temperature range, indicating that the ZIF-8 framework provides some protection for the enzyme molecules. Notably, CALB@L-His@ZIF-8 shows a relatively significant weight loss process in the 200-300°C range, suggesting the presence of a relatively large amount of organic components in the doped system, which may originate from the introduced L-His molecules and the embedded enzyme molecules. Furthermore, compared to pure ZIF-8, the framework decomposition temperature of L-His@ZIF-8 is slightly lower, indicating that the introduction of L-His alters the Zn content to some extent. 2+ The coordination environment between ZIF-8 and its ligands regulates the crystal structure of ZIF-8.

[0130] 2.2.6 CALB lipase Zeta potential

[0131] Zeta potential results showed that the surface potential of CALB@ZIF-8 decreased significantly from -0.9 mV of free CALB to -9.9 mV, while it rebounded to -3.8 mV after L-His doping. This indicates that the introduction of L-His significantly regulated the surface charge distribution of the immobilized system and improved the microenvironment around the enzyme.

[0132] 2.3 Enzymatic Properties Analysis

[0133] 2.3.1 Optimal temperature and temperature stability of immobilized lipase

[0134] The results showed that both free CALB and the two immobilized enzymes reached their maximum relative enzyme activity at 60°C. However, compared with the free enzyme, CALB@ZIF-8 and CALB@L-HIS@ZIF-8 still maintained higher residual activity in the high-temperature region (≥70°C), demonstrating that immobilization significantly improved the thermostability of the enzyme.

[0135] When both free and immobilized enzymes were incubated at 60°C, their relative enzyme activities gradually decreased with increasing treatment time (0-10 h), but the enzyme activity decayed more slowly after encapsulation. The free CALB enzyme activity decreased the fastest, reaching only 54.5% after 10 h; CALB@ZIF-8 activity was close to that of the free enzyme at 55.2% after 10 h; CALB@L-HIS@ZIF-8 maintained the highest level throughout the process, still at 68.6% after 10 h.

[0136] 2.3.2 Optimal pH and pH stability of immobilized lipase

[0137] The optimal pH for immobilized enzymes is concentrated at pH 8, and their activity remains more moderate in the pH range of 7-10. The L-HIS doped system exhibits higher relative enzyme activity under both acidic and alkaline conditions, indicating that it has an effective buffering and protective effect on the enzyme microenvironment.

[0138] When the free enzyme and the immobilized enzyme were incubated in a solution at pH 8 for 10 h, the free CALB decreased to 48.3% in 8-10 h; the CALB@ZIF-8 decreased to 58.8% in 10 h; and the L-HIS@ZIF-8@CALB remained at 70.2% in 10 h.

[0139] 2.3.3 Organic solvent tolerance of immobilized lipase

[0140] In polar solvents such as methanol, ethanol, and isopropanol, the activity of the free enzyme decreased significantly, while the immobilized enzyme retained approximately 80–90% of its activity, indicating that the encapsulation structure can reduce the impact of polar solvents on enzyme conformation. In the nonpolar solvent of n-hexane, both the free and immobilized enzymes exhibited high activity, with CALB@ZIF-8 showing the highest activity. This suggests that the hydrophobic environment helps stabilize the enzyme's spatial conformation and enhances substrate binding affinity, thereby improving catalytic efficiency. In contrast, the L-HIS doped system maintained similar or slightly higher activity in various solvents, indicating that it further improved the enzyme's flexibility and stability by regulating the hydrophilic / hydrophobic balance of the carrier microenvironment.

[0141] 2.3.4 Protein secondary structure analysis

[0142] Compared to free CALB, the secondary structure of the immobilized enzyme underwent significant changes. The β-sheet content in CALB@ZIF-8 decreased significantly, while the β-turn content increased significantly, indicating that the rigid structure of the enzyme molecule was disturbed during ZIF-8 growth and transitioned to a more flexible conformation. In contrast, after the introduction of L-His, the β-sheet content in CALB@L-HIS@ZIF-8 rebounded, while the β-turn remained at a high level, indicating that the presence of L-His mitigated the damage to the enzyme structure caused by MOF crystallization to some extent, making the enzyme conformation closer to its native state. Simultaneously, the α-helix content decreased slightly while the random coil remained relatively stable, indicating that the enzyme molecule only underwent local conformational rearrangement without significant denaturation. These results suggest that the introduction of L-His helps to improve the conformational flexibility of the enzyme while maintaining its structural stability, thereby facilitating substrate access to the active site and enhancing catalytic performance.

[0143] 2.3.5 Storage stability of immobilized lipase

[0144] CALB@ZIF-8 and CALB@L-His@ZIF-8 were stored at 4°C. During 60 days of storage, the relative enzyme activities of both immobilized lipases gradually decreased over time, but CALB@L-HIS@ZIF-8 showed a higher enzyme activity retention rate at all time points. The L-HIS-doped ZIF-8 carrier effectively slowed down enzyme activity decay, retaining approximately 69% of the relative enzyme activity after 60 days, significantly better than the 60% retention of the undoped system, indicating its significant advantage in long-term stability.

[0145] 2.3.6 Reusability of Immobilized Lipase

[0146] With increasing cycle number, the enzyme activity of CALB@ZIF-8 showed a significant downward trend. After 10 cycles and 10 reuses, the enzyme activity was still above 75%, which significantly improved the cycling stability of the immobilized lipase.

Claims

1. A method for preparing immobilized Candida antarcticis lipase, characterized in that, The steps include the following: (1) Mix 2-methylimidazole with deionized water to obtain a 2-methylimidazole solution; (2) Add Candida antarctica lipase solution to the 2-methylimidazole solution in (1) and mix well. This solution is called solution I. (3) Dissolve zinc acetate dihydrate in deionized water to obtain zinc acetate dihydrate solution; (4) Add the zinc acetate dihydrate solution from (3) to solution I, shake to mix, and then let it stand at room temperature to react; (5) After the reaction was completed, the precipitate was collected by centrifugation, washed with deionized water and anhydrous ethanol respectively, and dried under reduced pressure at room temperature to obtain CALB@ZIF-8 immobilized enzyme.

2. The method for preparing immobilized Candida antarcticis lipase as described in claim 1, characterized in that, (1) The mass-to-volume ratio of 2-methylimidazole to deionized water is (0.40-0.50) g: (12-18) mL.

3. The method for preparing immobilized Candida antarcticis lipase as described in claim 1, characterized in that, (2) The concentration of Candida antarcticis lipase solution is 1-2 mg / mL; the volume ratio of Candida antarcticis lipase solution to 2-methylimidazole solution is (12~18):(4~6) mL.

4. The method for preparing immobilized Candida antarcticis lipase as described in claim 1, characterized in that, In (3), the mass-to-volume ratio of zinc acetate dihydrate to deionized water is (0.16~0.20) g:(12~18) mL.

5. The method for preparing immobilized Candida antarcticis lipase as described in claim 1, characterized in that, (4) After shaking and mixing for 8-12s, let it stand at room temperature for 10-14h; the volume ratio of zinc acetate dihydrate solution to solution I is (12~18)mL:(16~24)mL.

6. The method for preparing immobilized Candida antarcticis lipase as described in claim 1, characterized in that, Centrifugation speed: 5000-7000 rpm; centrifugation time: 8-12 min; wash twice with deionized water and anhydrous ethanol respectively.

7. The method for preparing immobilized Candida antarcticis lipase as described in claim 1, characterized in that, Add L-histidine to the 2-methylimidazole solution in (1) in an amount of 10-30% of the mass of the 2-methylimidazole solution, and then proceed to (2).

8. The method for preparing immobilized Candida antarcticis lipase as described in claim 1, characterized in that, The steps include the following: (1) Mix 2-methylimidazole with deionized water at a mass-to-volume ratio of (0.40-0.50) g:(12-18) mL to obtain a 2-methylimidazole solution; (2) Add 1-2 mg / mL Candida antarcticis lipase solution to the 2-methylimidazole solution in (1) and mix well; the volume ratio of Candida antarcticis lipase solution to 2-methylimidazole solution is (12~18):(4~6) mL; denoted as solution I; (3) Dissolve zinc acetate dihydrate in deionized water to obtain zinc acetate dihydrate solution; the mass-to-volume ratio of zinc acetate dihydrate to deionized water is (0.16-0.20) g:(12-18) mL; (4) Add the zinc acetate dihydrate solution from (3) to solution I, shake and mix for 8-12 seconds, and then let it stand at room temperature for 10-14 hours. The volume ratio of zinc acetate dihydrate solution to solution I is (12~18) mL: (16~24) mL. (5) After the reaction was completed, the precipitate was collected by centrifugation at 5000-7000 rpm for 8-12 min, washed twice with deionized water and anhydrous ethanol, and finally dried under reduced pressure at room temperature to obtain CALB@ZIF-8 immobilized enzyme.

9. The method for preparing immobilized Candida antarcticis lipase as described in claim 1, characterized in that, The steps include the following: (1) Mix 2-methylimidazole with deionized water at a mass-to-volume ratio of (0.40-0.50) g:(12-18) mL to obtain a 2-methylimidazole solution; then add L-histidine in an amount of 10-30% of the mass of the 2-methylimidazole solution; this is referred to as solution II. (2) Add 1-2 mg / mL Candida antarcticis lipase solution to the solution obtained in (1) and mix well; the volume ratio of Candida antarcticis lipase solution to 2-methylimidazole solution is (12~18):(4~6) mL; denoted as solution I; (3) Dissolve zinc acetate dihydrate in deionized water to obtain zinc acetate dihydrate solution; the mass-to-volume ratio of zinc acetate dihydrate to deionized water is (0.16-0.20) g:(12-18) mL; (4) Add the zinc acetate dihydrate solution from (3) to solution I, shake and mix for 8-12 seconds, and then let it stand at room temperature for 10-14 hours. The volume ratio of zinc acetate dihydrate solution to solution I is (12~18) mL: (16~24) mL. (5) After the reaction was completed, the precipitate was collected by centrifugation at 5000-7000 rpm for 8-12 min, washed twice with deionized water and anhydrous ethanol, and finally dried under reduced pressure at room temperature to obtain CALB@ZIF-8 immobilized enzyme.

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