Immobilized enzyme Fe3O4-Rha as well as preparation method and application thereof

Abstract

The invention discloses an immobilized enzyme Fe3O4-Rha as well as a preparation method and application thereof, and belongs to the technical field of immobilized enzymes. The Fe3O4 nano particles provided by the invention have good superparamagnetism; the immobilized enzyme Fe3O4-Rha prepared by taking the immobilized enzyme Fe3O4-Rha as the carrier has good magnetic responsiveness, the optimum pH is 6.0, the optimum temperature is 40 DEG C, and the tolerance to an organic solvent is superior to that of a free enzyme; naringin can be catalyzed to be converted into cherry glycoside, and the conversion rate reaches 98.01%; the reusability is good, and the relative enzyme activity is 61.34% after the enzyme is recycled for 5 times; after the system is amplified, the immobilized enzyme Fe3O4-Rha can still realize the efficient conversion of the naringin; the provided preparation method is simple and easy to operate, the prepared Fe3O4 nanoparticles are good in dispersity, and the immobilization efficiency of alpha-L-rhamnosidase is 49.40%; the method has low requirements on equipment, does not use toxic reagents, does not generate toxic and harmful substances, and is green and environment-friendly.

Description

Technical Field

[0001] The present invention relates to the technical field of immobilized enzymes, and particularly relates to an immobilized enzyme Fe3O4-Rha and its preparation method and application. Background Art

[0002] Enzymes are natural biocatalysts, which have the advantages of high specificity, high activity, high catalytic efficiency, mild action conditions, and extremely few by-products. However, free enzymes generally have limitations such as being sensitive to harsh environments, being prone to denaturation, having poor stability, being difficult to separate from solutions, having a long processing time, being difficult to recycle products, and being difficult to reuse, resulting in high usage costs of free enzyme systems and limited widespread applications. Immobilizing enzymes on ideal carriers to prepare immobilized enzymes is an effective strategy to solve the above problems. Immobilized enzymes have the advantages of good stability, low cost, easy separation, and recyclability, and have good application prospects.

[0003] α-L-rhamnosidase is widely present in fungi, mammalian tissues, plants, and bacteria, and is an important glycoside hydrolase with wide industrial applications. By cleaving α-1,2, α-1,3, α-1,4, or α-1,6 glycosidic bonds, α-L-rhamnosidase can effectively and specifically cleave various natural compounds containing non-reducing terminal L-rhamnose residues. For example, after specifically removing the α-L-rhamnosyl group of a compound, flavonoid compounds such as rutin and naringin can be respectively converted into monoflavonoid glycoside products such as isoquercetin and prunasin. The contents of these products in nature are relatively low, but compared with flavonoid compounds such as rutin and naringin, the bioavailability and biological activities of these products are greatly enhanced. In addition, some α-L-rhamnosidases can catalyze the reverse hydrolysis synthesis of rhamnosides with rhamnose as the donor. Summary of the Invention

[0004] The purpose of the present invention is to provide an immobilized enzyme Fe3O4-Rha and its preparation method and application, and to provide a new immobilized enzyme of α-L-rhamnosidase with high immobilization efficiency, high activity, good stability, and magnetic response.

[0005] To achieve the above purpose, the present invention provides a preparation method of an immobilized enzyme Fe3O4-Rha, including the following steps: S1. Prepare hydrophobic Fe3O4 nanoparticles. Stir and mix iron acetylacetonate, 1,2-dodecanediol, oleic acid, oleylamine, and benzyl ether under a protective atmosphere, and then react under an air atmosphere to obtain a black mixture. After cooling to room temperature, add anhydrous ethanol and centrifuge. Add oleic acid, oleylamine, and n-hexane to the precipitate in sequence, mix well and centrifuge to take the supernatant, and then add anhydrous ethanol and centrifuge to take the precipitate, which is the hydrophobic Fe3O4 nanoparticles; S2. Phase transfer the hydrophobic Fe3O4 nanoparticles prepared in S1. Disperse the hydrophobic Fe3O4 nanoparticles in tetrahydrofuran to obtain a Fe3O4 nanoparticle dispersion. Add tetrahydrofuran to 3,4-dihydroxycinnamic acid. After dissolution, add dropwise the prepared Fe3O4 nanoparticle dispersion and react under a protective atmosphere. After the reaction, cool to room temperature, add NaOH solution and centrifuge. The precipitate is the hydrophilic Fe3O4 nanoparticles. S3. Activate the hydrophilic Fe3O4 nanoparticles prepared in S2. Disperse the hydrophilic Fe3O4 nanoparticles prepared in S2 in ultrapure water, add a crosslinking agent and PBS buffer in sequence, shake and incubate, then perform magnetic separation to remove the crosslinking agent to obtain the activated Fe3O4 nanoparticles. S4. Disperse the activated Fe3O4 nanoparticles obtained in S3 in PBS buffer to obtain an activated Fe3O4 nanoparticle PBS dispersion. Add α-L-rhamnosidase Rha and perform a curing reaction on a shaker. After the reaction, perform magnetic separation to obtain the immobilized enzyme Fe3O4-Rha.

[0006] Preferably, in S1, the molar ratio of iron acetylacetonate: 1,2-dodecanediol: oleic acid: oleylamine: benzyl ether is 1:5:3:3:53; the stirring and mixing condition is to stir and react at 200 °C for 2 h; the reaction condition under an air atmosphere is 300 °C and the reaction is for 1 h.

[0007] Preferably, in S1, the volume ratio of the added black mixture to absolute ethanol is 1:2 - 3; the volume ratio of the supernatant to absolute ethanol is 1:2 - 3; the volume ratio of oleic acid: oleylamine: n-hexane is 1:1:200.

[0008] Preferably, in S2, the mass-volume ratio of the hydrophobic Fe3O4 nanoparticles: tetrahydrofuran: 3,4-dihydroxycinnamic acid is 20 mg: 11 mL: 50 mg; 3,4-dihydroxycinnamic acid is dissolved in tetrahydrofuran under a protective atmosphere; the reaction condition under the protective atmosphere is to react at 50 °C for 3 h.

[0009] Preferably, in S2, the concentration of the NaOH solution is 0.5 M; the volume ratio of the addition amount of the NaOH solution to the volume of the nanoparticle mixed solution is 1:22.

[0010] Preferably, in S3, the crosslinking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1.2:1; when activating the Fe3O4 nanoparticles, the mass-volume ratio of the hydrophilic Fe3O4 nanoparticles: ultrapure water: crosslinking agent: PBS buffer is 10 mg: 1 mL: 4.4 mL: 4.4 mL.

[0011] Preferably, the conditions for oscillating incubation in S3 are room temperature, 150 - 200 rpm, and oscillating incubation for 20 - 40 min; the crosslinking agent is removed by washing with PBS buffer at pH 7.0.

[0012] Preferably, the mass ratio of the activated Fe3O4 nanoparticles to α-L-rhamnosidase Rha in S4 is 1:2; the curing reaction conditions are 4°C, 150 - 200 rpm, and 2 - 5 h.

[0013] An immobilized enzyme Fe3O4-Rha prepared by the method for preparing an immobilized enzyme Fe3O4-Rha as described above.

[0014] An application of the immobilized enzyme Fe3O4-Rha as described above in the preparation of a new compound, where the new compound is achieved by cleaving the α-L-rhamnoside bond.

[0015] Therefore, an immobilized enzyme Fe3O4-Rha provided by the present invention, its preparation method and application have the following specific technical effects: (1) Fe3O4 nanoparticles with good morphology and good water dispersibility were successfully prepared by the method provided by the present invention, and α-L-rhamnosidase was successfully immobilized on the Fe3O4 nanoparticles. When the mass ratio of the α-L-rhamnosidase addition amount to the Fe3O4 nanoparticles is 2:1, the activation time is 0.5 h, and the immobilization efficiency is the highest at 49.40% when curing at 4°C and pH 7.0 for 4 h; (2) The Fe3O4 nanoparticles prepared by the present invention have good superparamagnetism; the immobilized enzyme Fe3O4-Rha prepared with this as the carrier has good magnetic responsiveness and superparamagnetism, the optimum pH is 6.0, the optimum temperature is 40°C, and its tolerance to organic solvents is better than that of the free enzyme, especially ethanol and isopropanol; (3) The immobilized enzyme Fe3O4-Rha prepared by the present invention can be used for the efficient biotransformation of naringin, and the conversion rate can reach 98.01%; it has good reusability, and after being recycled 5 times, the relative enzyme activity is 61.34%; the immobilized enzyme Fe3O4-Rha can still well carry out the transformation of naringin after being scaled up; (4) The preparation method provided by the present invention is simple and easy to operate, has low requirements for equipment, does not use toxic reagents, and no toxic and harmful substances are generated, which is green and environmentally friendly.

[0016] The technical solutions of the present invention will be further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments of the present invention. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 are the SDS-PAGE gel electrophoresis and protein immunoblotting results in Example 1 of the present invention; where a is the SDS-PAGE gel electrophoresis diagram; b is the protein immunoblotting result; Figure 2 is the circular dichroism spectrum at room temperature in Example 1 of the present invention; Figure 3 is the circular dichroism spectrum in the range of 25°C to 85°C in Example 1 of the present invention; Figure 4 is the TEM photograph in Example 2 of the present invention; where a is the hydrophobic Fe3O4 nanoparticles; b is the hydrophilic Fe3O4 nanoparticles; Figure 5 is the protein standard curve in Example 2 of the present invention; Figure 6 is the influence of the enzyme addition amount on the immobilization efficiency in Example 2 of the present invention; Figure 7 is the influence of the activation time on the immobilization efficiency in Example 2 of the present invention; Figure 8 is the influence of the immobilization time on the immobilization efficiency in Example 2 of the present invention; Figure 9 is the influence of the immobilization temperature on the immobilization efficiency in Example 2 of the present invention; Figure 10 is the influence of pH on the immobilization efficiency in Example 2 of the present invention; Figure 11 is the investigation result of the magnetic response ability of the immobilized enzyme Fe3O4-Rha in Effect Test 1 of the present invention; where a is the solution of the immobilized enzyme Fe3O4-Rha without an external magnetic field; b is the magnetic separation of the immobilized enzyme Fe3O4-Rha under an external magnetic field; c is the immobilized supernatant after magnetic separation; Figure 12 is the transmission electron microscope photograph of the immobilized enzyme Fe3O4-Rha in Effect Test 2 of the present invention; Figure 13 is the SDS-PAGE electrophoresis result in Effect Test 3 of the present invention; Figure 14It is the Fourier transform infrared spectrum of the immobilized enzyme Fe3O4-Rha in Test 4 of the effects of the present invention; where a is Fe3O4; b is α-L-rhamnosidase Rha; c is the immobilized enzyme Fe3O4-Rha; Figure 15 It is the hysteresis curve of Fe3O4 nanoparticles and the immobilized enzyme Fe3O4-Rha in Test 5 of the effects of the present invention; Figure 16 It is the thermogravimetric analysis curve of Fe3O4 nanoparticles and the immobilized enzyme Fe3O4-Rha in Test 6 of the effects of the present invention; Figure 17 It is the standard curve of prunasin in Test 7 of the effects of the present invention; Figure 18 It is the effect of pH on the activities of free α-L-rhamnosidase Rha and the immobilized enzyme Fe3O4-Rha in Test 7 of the effects of the present invention; Figure 19 It is the effect of temperature on the activities of free α-L-rhamnosidase Rha and the immobilized enzyme Fe3O4-Rha in Test 7 of the effects of the present invention; Figure 20 It is the tolerance of free α-L-rhamnosidase Rha and the immobilized enzyme Fe3O4-Rha to organic solvents in Test 7 of the effects of the present invention; Figure 21 It is the effect of the concentration of the immobilized enzyme Fe3O4-Rha on the conversion rate in Test 7 of the effects of the present invention; Figure 22 It is the effect of the enzymatic reaction time of the immobilized enzyme Fe3O4-Rha on the conversion rate in Test 7 of the effects of the present invention; Figure 23 It is the effect of the substrate concentration on the conversion rate in Test 7 of the effects of the present invention; Figure 24 It is the HPLC chromatogram of the biotransformation of naringin by Fe3O4-Rha in Test 1 of the effects of the present invention; where A is the HPLC chromatogram of a mixture of naringin and prunasin standards (molar ratio 1:1); B is the HPLC chromatogram of the control group; C is the HPLC chromatogram of the free enzyme group; D is the HPLC chromatogram of the immobilized enzyme group; Figure 25 It is the result of the investigation of the reusability of the immobilized enzyme Fe3O4-Rha in Test 7 of the effects of the present invention; Figure 26 It is the conversion of naringin by the immobilized enzyme Fe3O4-Rha in an enlarged system in Test 7 of the effects of the present invention. Detailed implementation mode

[0019] The technical solution of the present invention will be further described below with reference to the drawings and embodiments.

[0020] In order to make the objectives, technical solutions and advantages of the present application clearer, more thorough and complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all descriptions of embodiments, aiming to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs.

[0021] The instrument and equipment, reagent materials used in the embodiments are all obtained through commercial channels; the method steps not described in detail are all conventional technical means in the art.

[0022] Example 1 Prepare α-L-rhamnosidase Rha as follows: S11. Thaw the Escherichia coli engineering strain BL21(DE3) / pET-28a- HFM-RhaA (generously donated by the laboratory of Teacher Li Binchun, Institute of Biotechnology, Shanxi University) on ice. After thawing, pipette 50 μL into 5 mL of 2×YT medium containing 5 μL of 50 mg / mL Kana, and culture the strain at 37 °C and 180 rpm for 12 h to resuscitate the strain. Then, use the same method, medium and ratio to perform flask scale-up culture.

[0023] S12. When the OD 600 of the bacterial liquid in the flask is about 1.8, add IPTG with a final concentration of 0.5 mM, continue to culture at 16 °C and 180 rpm for 12 h, centrifuge at 4 °C and 8000 rpm for 10 min, discard the supernatant, resuspend the cells with 50 mM PB buffer at pH 8.0, and then ultrasonically disrupt for 30 min. The power of the ultrasonic disruptor is set to 30%, and the mode is ultrasonic for 2 s and intermittent for 3 s.

[0024] S13. Centrifuge at 4 °C and 12000 rpm for 40 min, take the supernatant and perform affinity purification using a nickel column to obtain α-L-rhamnosidase Rha. Perform SDS-PAGE gel electrophoresis and Western blot analysis on the purified α-L-rhamnosidase Rha. The results are as Figure 1 shown, indicating that high-purity α-L-rhamnosidase Rha containing His tag was successfully prepared.

[0025] Another purified α-L-rhamnosidase Rha was dialyzed against salt using 2 L of 50 mM PB buffer at pH 8.0 to obtain Rha enzyme, which was then diluted to a certain concentration with PB buffer. The Rha enzyme was analyzed at room temperature (25 °C) using a circular dichroism spectrometer: A cuvette with an optical path length of 0.05 cm was selected and scanned in the wavelength range of 190 - 260 nm. Then, the CDNN software was used to analyze the secondary structure of the Rha enzyme. The results are as Figure 2 shown. The secondary structure of α-L-rhamnosidase Rha includes 11.2% α-helix, 41.6% β-sheet (parallel and anti-parallel), 17.5% β-turn, and 32.7% random coil.

[0026] Then, the Rha enzyme diluted to a certain concentration with PB buffer was used to collect the CD spectra of the Rha enzyme by a circular dichroism spectrometer at a frequency of scanning once every 5 °C increase in the range of 25 °C - 85 °C. According to the CD spectral data, the temperature change curve of the Rha enzyme was plotted. The results are as Figure 3 shown. In the range of 25 °C - 55 °C, the CD spectra of the Rha enzyme basically completely overlap, indicating that the Rha enzyme has good stability in this temperature range. In the range of 75 °C - 85 °C, the CD spectra of the Rha enzyme have changed significantly, indicating that the secondary structure of the Rha enzyme has changed at high temperatures.

[0027] Example 2 To prepare an immobilized enzyme Fe3O4-Rha, the specific steps are as follows: S21. Synthesis of hydrophobic Fe3O4 nanoparticles. 0.3532 g (1 mmol) of iron(III) acetylacetonate and 1.0117 g (5 mmol) of 1,2-dodecanediol accurately weighed were added into a 100 mL three-necked flask. Then, 1.022 mL (3 mmol) of oleic acid, 0.99 mL (3 mmol) of oleylamine, and 10 mL (53 mmol) of benzyl ether were successively added using a pipette. Then, under a nitrogen flow, it was stirred and mixed at 200 °C for 2 h using a digital display intelligent temperature-controlled magnetic stirrer.

[0028] After the reaction ended, the nitrogen was turned off. Then, the temperature of the digital display intelligent temperature-controlled magnetic stirrer was set to 300 °C, and the mixture was stirred and reacted at 300 °C for 1 h to obtain a black mixture. After cooling to room temperature, 30 mL of absolute ethanol was added, and after mixing, it was centrifuged at 12000 rpm for 20 min. The supernatant was discarded. 25 μL of oleic acid, 25 μL of oleylamine, and 5 mL of n-hexane were successively added to the precipitate. After mixing, it was centrifuged at 6000 rpm for 10 min. The supernatant was transferred to a new centrifuge tube, 10 mL of absolute ethanol was added, and it was centrifuged at 12000 rpm for 20 min. The obtained black precipitate was dried with nitrogen to obtain 78 mg of hydrophobic Fe3O4 nanoparticles, which were stored at 4 °C.

[0029] The obtained hydrophobic Fe3O4 nanoparticles were configured into a 10 mg / mL solution with n-hexane and detected using a transmission electron microscope (JEM2100PLUS). The results are as Figure 4 shown in a of [reference], and the magnetic nanoparticles have good morphology and uniform distribution.

[0030] S22. Phase transfer the hydrophobic Fe3O4 nanoparticles obtained in step S21. Accurately weigh 20 mg of the hydrophobic Fe3O4 nanoparticles obtained in step S21 and disperse them in 1 mL of tetrahydrofuran to obtain a Fe3O4 nanoparticle dispersion.

[0031] Add 10 mL of tetrahydrofuran to 50 mg of 3,4-dihydroxycinnamic acid in a fume hood. Under nitrogen conditions, wait until the temperature of the stirrer rises to 50 °C, and then dropwise add the prepared Fe3O4 nanoparticle dispersion. Keep reacting for 3 h under nitrogen and 50 °C conditions. After the reaction, cool to room temperature, then add 500 μL of 0.5 M NaOH, mix well, and centrifuge at 3000 rpm for 1 min. The precipitate is the hydrophilic Fe3O4 nanoparticles. Disperse the Fe3O4 nanoparticles in ultrapure water and store them at 4 °C.

[0032] The obtained ultrapure water dispersion of Fe3O4 nanoparticles (at a concentration of 10 mg / mL) was detected using a transmission electron microscope (JEM2100PLUS). The results are as Figure 4 shown in b of [reference], and the magnetic nanoparticles have good morphology and uniform distribution.

[0033] S23. Carry out an activation reaction on the hydrophilic Fe3O4 nanoparticles obtained in step S22. Add 120 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 0.1 moL / L), 100 μL of N-hydroxysuccinimide (NHS, 0.1 moL / L) to 50 μL of the ultrapure water dispersion of 10 mg / mL Fe3O4 nanoparticles, and then add 220 μL of pH 7.0 PBS buffer. Incubate with shaking at room temperature and 175 rpm for 30 min, then perform magnetic separation, and wash 3 times with 500 μL of pH 7.0 PBS buffer to remove the excess cross-linking agents (EDC and NHS), thus obtaining 0.5 mg of the activated Fe3O4 nanoparticles. Redisperse the activated Fe3O4 nanoparticles in 490 μL of pH 7.0 PBS buffer to obtain an activated Fe3O4 nanoparticle PBS dispersion.

[0034] S24. Draw a protein standard curve for the determination of the immobilization amount of α-L-rhamnosidase later. Use bovine serum albumin (BSA) at 0.5 mg / mL as the standard protein. Add 0, 0.5, 1, 2, 4, 6, 8, 10 μL of the standard protein into the 96-well plate respectively. For those less than 10 μL, make up to 10 μL with ultrapure water. The protein concentration gradients are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL respectively. Then add 200 μL of Coomassie Brilliant Blue G-250 staining solution (including 50 mg of Coomassie Brilliant Blue G-250, 25 mL of 95% ethanol, 50 mL of 85% phosphoric acid, and made up to 500 mL with ultrapure water) to each well. After mixing, incubate at room temperature for 12 min. Then use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value at 595 nm to obtain the protein standard curve. As Figure 5 shown, y = 1.1822x + 0.0186, R 2 = 0.9911, with a good correlation and can be used as the standard curve for determining the immobilization amount of Rha.

[0035] S25. Optimize the immobilization parameters.

[0036] 1) Effect of enzyme addition amount on the immobilization efficiency.

[0037] Add α-L-rhamnosidase Rha to the activated Fe3O4 nanoparticle PBS dispersion prepared in step S23. The addition amounts of Rha are 0.25, 0.5, 1, 1.5, 2 mg respectively. Make up to 800 μL with pH 7.0 PBS buffer and immobilize at 4 °C on a shaker at 175 rpm for 4 h. Set 3 parallels for each gradient. After immobilization, perform magnetic separation, wash 3 times with pH 7.0 PBS buffer, collect the supernatant and washing solution mixture, use an ELISA reader to detect the absorbance value at 595 nm of the supernatant respectively, substitute it into the protein standard curve obtained in step S24 to obtain the enzyme concentration in the supernatant, and use the formula immobilization efficiency = enzyme amount in the supernatant / enzyme amount added during immobilization × 100% to obtain the immobilization efficiency. The results are as Figure 6 shown. When the addition amount of Rha is 0.25 mg, the immobilization efficiency is the highest, which is 60.65%. Followed by when the addition amount of Rha is 1 mg, the immobilization efficiency is 49.73%. However, compared with the addition amount of 1 mg of Rha, the amount of enzyme immobilized in the 0.25 mg Rha addition group is less. Therefore, select 1 mg of Rha enzyme addition amount as the optimal enzyme addition amount for immobilization.

[0038] 2) Effect of activation time on the immobilization efficiency.

[0039] To the 50 μL ultrapure water dispersion of Fe3O4 nanoparticles (10 mg / mL) obtained in step S22, 120 μL of EDC (0.1 moL / L) and 100 μL of NHS (0.1 moL / L) were successively added. Then, 220 μL of pH 7.0 PBS buffer was added, and the mixture was incubated with shaking at room temperature and 175 rpm for 0.5, 1, 2, 3, 4, and 5 h. Three parallels were set for each gradient. Then, magnetic separation was performed, and the mixture was washed with pH 7.0 PBS buffer to remove the excess cross-linking agent. The activated Fe3O4 nanoparticles were then redispersed in 490 μL of pH 7.0 PBS buffer. Then, 1 mg of α-L-rhamnosidase Rha was added, and the volume was made up to 800 μL with pH 7.0 PBS buffer, and the mixture was incubated (immobilized) at 4 °C and 175 rpm for 4 h. After the immobilization was completed, magnetic separation was performed, and the mixture was washed 3 times with pH 7.0 PBS buffer. The supernatant and washing solution mixture were collected, and the immobilization efficiency was calculated using the method in 1). The results are as Figure 7 shown. The immobilization efficiencies after activating Fe3O4 nanoparticles for 0.5, 1, 2, 3, 4, and 5 h were 48.58%, 48.99%, 48.71%, 49.34%, 45.92%, and 48.07% respectively. It can be seen that during immobilization, the immobilization efficiency of activating for 0.5 h has little difference from that of other groups. Therefore, 0.5 h was selected as the optimal time for activating Fe3O4 nanoparticles.

[0040] 3) Effect of immobilization time on immobilization efficiency.

[0041] To the 490 μL of PBS buffer containing activated Fe3O4 nanoparticles prepared in step S23, 1 mg of α-L-rhamnosidase Rha was added, and the volume was made up to 800 μL with pH 7.0 PBS buffer, and the mixture was immobilized at 4 °C and 175 rpm for 1, 2, 3, 4, and 5 h respectively. Three parallels were set for each gradient. After the immobilization was completed, magnetic separation was performed, and the mixture was washed 3 times with pH 7.0 PBS buffer. The supernatant and washing solution mixture were collected, and the immobilization efficiency was calculated using the method in 1). The results are as Figure 8 shown. As the immobilization time increased, the immobilization efficiency first increased and then decreased. The immobilization efficiency was the best after 4 h of immobilization, which was 46.49%. Therefore, 4 h was selected as the optimal time for immobilization.

[0042] 4) Effect of immobilization temperature on immobilization efficiency.

[0043] Add 1 mg of α-L-rhamnosidase Rha to 490 μL of PBS buffer containing activated Fe3O4 nanoparticles, and then make up to 800 μL with PBS buffer at pH 7.0. Then, immobilize at 175 rpm on a shaker at 4 °C, 20 °C, 30 °C, 35 °C, and 40 °C for 4 h respectively. Set 3 parallels for each gradient. After immobilization, perform magnetic separation, wash 3 times with PBS buffer at pH 7.0, collect the supernatant and washing solution mixture, and calculate the immobilization efficiency using the method in 1). The results are as Figure 9 shown. When the temperature is 4 °C and 35 °C, the immobilization efficiency is relatively high, 47.20% and 44.86% respectively. Figure 6 b of Figure 6 shows the state of each group of solutions after immobilization. It can be seen that when immobilizing at 35 °C, there are obvious flocculent precipitates after immobilization. It may be that flocculent precipitates are generated during the immobilization of the enzyme. Therefore, 4 °C is selected as the optimal temperature for immobilization.

[0044] 5) Effect of pH on immobilization efficiency.

[0045] Add 1 mg of α-L-rhamnosidase Rha to 490 μL of PBS buffer containing activated Fe3O4 nanoparticles, make up to 800 μL with PBS buffer of different pH values, and immobilize on a shaker at 4 °C and 175 rpm for 4 h. The pH values of the PBS buffer are 5.0, 6.0, 7.0, 8.0, and 9.0 respectively. Set 3 parallels for each gradient. After immobilization, perform magnetic separation, wash 3 times with PBS buffer at pH 7.0, collect the supernatant and washing solution mixture, and calculate the immobilization efficiency using the method in 1). The results are as Figure 10 shown. When immobilizing, when the pH is 7.0, the immobilization efficiency is the highest, 49.40%. Therefore, 7.0 is selected as the optimal pH for immobilization.

[0046] The optimal immobilization parameters obtained are: the enzyme addition amount is 1 mg, the activation time is 0.5 h, the immobilization time is 4 h, the temperature is 4 °C, and the pH is 7.0.

[0047] Effect test 1 Examine the magnetic response ability of the immobilized enzyme Fe3O4-Rha prepared in Example 2. Place 1 mL of the immobilized enzyme solution in a small glass bottle, place a magnet with a magnetic field strength of 4 T outside the bottle wall, and separate the supernatant after 20 s and place it in another clean small glass bottle.

[0048] The results are as Figure 11 shown. As can be seen from Figure 11 a of Figure 11 , when there is no external magnetic field, the immobilized enzyme Fe3O4-Rha is evenly dispersed in the solution. As can be seen from Figure 11As can be seen from b, when there is a magnet, the immobilized enzyme Fe3O4-Rha can aggregate to the position of the magnet in 19 seconds, and the time is less than 20 s, indicating that the immobilized enzyme Fe3O4-Rha has good magnetic responsiveness. Figure 11 c in the figure is the supernatant after magnetic separation of the immobilized enzyme Fe3O4-Rha solution, and it can be observed that the supernatant has almost no color.

[0049] Effect test 2 The immobilized enzyme Fe3O4-Rha prepared in Example 2 was analyzed by transmission electron microscopy (TEM, model:), and the results are as Figure 12 shown. A white substrate can be observed around the Fe3O4 nanoparticles, which may be due to the immobilization of Rha enzyme on the surface of the Fe3O4 nanoparticles.

[0050] Effect test 3 The immobilized enzyme Fe3O4-Rha prepared in Example 2 was analyzed by protein gel as follows: Take 10 µL of 5×Loading buffer and mix it with 40 µL of the supernatant and washing solution mixture obtained by immobilization using the optimal immobilization parameters obtained in Example 2. Then take 10 µL of 5×Loading buffer and mix it with 40 µL of the solution of the control group. The control group is a PBS buffer solution containing the same amount of Rha as the enzyme added at the beginning of immobilization (the volume of the PBS buffer solution is the same as the total volume of the supernatant and washing solution mixture). Then boil the samples in a metal bath at 100 °C for 6 min to obtain two protein samples, and then perform SDS-PAGE electrophoresis. After the electrophoresis is completed, the protein gel is stained with Coomassie Brilliant Blue R-250 staining solution and then decolorized with a decolorizing solution to obtain a protein gel with clear bands. Finally, Image J is used for gray value analysis.

[0051] The results are as Figure 13 shown. Through gray analysis by Image J, the gray value of the immobilized supernatant accounts for about 56.35% of the control group. The gray analysis results show that after immobilization, the Rha enzyme was successfully immobilized on the Fe3O4 nanoparticles.

[0052] Effect test 4 The immobilized enzyme Fe3O4-Rha prepared in Example 2 was analyzed by Fourier transform infrared spectroscopy (FTIR) as follows: The hydrophilic Fe3O4 nanoparticles, Rha particles and Fe3O4-Rha particles prepared in Example 2 were dried and then detected by a Fourier transform infrared spectrometer (Thermo Nicolet iS5), and the scanning range was 4000~400 cm -1 .

[0053] The results are asFigure 14 As shown, in the figure, a is the infrared spectrum of Fe3O4 nanoparticles. At 585 cm -1 , it is the characteristic signal of the Fe-O bond. At 1406 cm -1 and 924 cm -1 , they are the characteristic signals of the hydroxyl groups (-OH) on the carboxyl groups on the surface of Fe3O4 nanoparticles. In the figure, b is the infrared spectrum of rhamnosidase. At 1650 cm -1 and 1540 cm -1 , they are the characteristic signals of amide I band and amide II band respectively. In the figure, c is the infrared spectrum of the immobilized enzyme Fe3O4-Rha. The characteristic signal of the Fe-O bond was observed at 586 cm -1 ; at the same time, the characteristic signals of the amide I band and amide II band of rhamnosidase could be detected at 1637 cm -1 and 1542 cm -1 ; while the characteristic signal intensity of the hydroxyl groups (-OH) on the carboxyl groups on the surface of Fe3O4 nanoparticles decreased significantly at 1400 cm -1 , and the signal disappeared at 924 cm -1 , indicating that a chemical reaction occurred between the carboxyl groups on the surface of Fe3O4 nanoparticles and the amino groups on rhamnosidase. It shows that α-L-rhamnosidase Rha has been successfully immobilized on Fe3O4 nanoparticles.

[0054] Effect Test 5 Magnetic detection (SQUID) was performed on the immobilized enzyme Fe3O4-Rha prepared in Example 2 as follows: The superconducting quantum interference device (SQUID) was used to detect the magnetic properties of the Fe3O4 nanoparticles and Fe3O4-Rha particles prepared in Example 2. The test temperature was 298 K, and the change range of the magnetic field strength was -70000~70000 Oe.

[0055] The results are as Figure 15 shown. The saturation magnetization intensity (Ms) of Fe3O4 nanoparticles was 24.29 emu / g, and the saturation magnetization intensity (Ms) of Fe3O4-Rha was 21.86 emu / g. It can be seen that after immobilization, the saturation magnetization intensity of Fe3O4-Rha decreased slightly, which may be because a layer of Rha enzyme was bound to the surface of Fe3O4 nanoparticles, resulting in magnetic loss. When the applied magnetic field (H) was 0, the remanent magnetization intensity (Mr) and coercivity (Hc) were both close to 0, proving that both Fe3O4 nanoparticles and Fe3O4-Rha had good superparamagnetism.

[0056] Effect Test 6 Thermogravimetric analysis (TGA) was performed on the immobilized enzyme Fe3O4-Rha prepared in Example 2 as follows: The Fe3O4 nanoparticles and Fe3O4-Rha nanoparticles prepared in Example 2 were analyzed using a thermogravimetric analyzer (STA 449 F5) under an air atmosphere. The test temperature range was 25°C to 800°C, and the heating rate was 10°C / min.

[0057] The results are as Figure 16 shown. From 25°C to 800°C, weight loss occurred for both the Fe3O4 nanoparticles and the immobilized enzyme Fe3O4-Rha. At 800°C, the remaining weight of the Fe3O4 nanoparticles was approximately 64.42%. Under the same conditions, the remaining weight of Fe3O4-Rha was approximately 57.29%. By calculation, the loading amount of Rha enzyme on the Fe3O4 nanoparticles was 124.43 mg / g, indicating that the Rha enzyme was successfully immobilized on the Fe3O4 nanoparticles.

[0058] Effect Test 7 The enzymatic properties of the immobilized enzyme Fe3O4-Rha prepared in Example 2 were investigated as follows: (1) The ability of Fe3O4-Rha to biotransform naringin was detected by high performance liquid chromatography (HPLC).

[0059] After mixing 180 μL of 50 mM phosphate buffer at pH 6.0 with 10 μL of 20 mM naringin (dissolved in DMSO), it was incubated at 40°C for 2 min, with 3 parallels in each group. 10 μL of free enzyme or immobilized enzyme Fe3O4-Rha was added, and the reaction was carried out in a constant temperature mixer for 1 h, with the rotation speed of the constant temperature mixer set at 800 rpm. After the reaction ended, 800 μL of methanol (chromatographically pure) was immediately added to terminate the reaction. It was centrifuged at 13000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm organic filter membrane. Finally, it was detected using high performance liquid chromatography. The specific detection conditions were as follows: The mobile phase and ratio were 0.5% glacial acetic acid:acetonitrile volume ratio = 77:23; the flow rate was 1.0 mL / min, and the injection volume was 10 μL; the peak area of prunin was detected at 283 nm using a chromatographic column (Hypersil OSD2-C18) at room temperature, and the detection time was set to 10 min. The conversion rate was calculated using the following formula: Conversion rate = product prunin concentration (mM) / substrate naringin initial concentration (mM) × 100%.

[0060] Enzyme activity assay method: Under the optimal reaction conditions, the amount of enzyme required to generate 1 μmol of prunin per minute was defined as 1 enzyme activity unit (U).

[0061] The specific activity of the free enzyme Rha was 2.9 U / mg, and the specific activity of the immobilized enzyme Fe3O4-Rha was 0.0483 U / mg.

[0062] (2)Characterize the enzymatic properties of Fe3O4-Rha.

[0063] 1) Plot the standard curve of prunasin. Prepare a 20 mM prunasin stock solution with DMSO as the solvent, and then dilute the prunasin stock solution to different concentrations with methanol (chromatographic grade): 0 mM, 0.02 mM, 0.04 mM, 0.06 mM, 0.08 mM, 0.1 mM, 0.12 mM, 0.14 mM, 0.16 mM, 0.18 mM, 0.2 mM. After filtering with a 0.22 µm organic filter membrane, analyze by high performance liquid chromatography. The specific detection conditions are as follows: the mobile phase is 0.5% glacial acetic acid: acetonitrile volume ratio = 77:23; the flow rate is 1.0 mL / min, and the injection volume is 10 µL; use a chromatographic column (Hypersil OSD2-C18) to detect the peak area of prunasin at 283 nm at room temperature, and set the detector time to 10 min. Using the prunasin concentration and the corresponding peak area as the abscissa and ordinate respectively, plot the graph with Origin to obtain the standard curve of prunasin. As Figure 17 shown, the standard curve of prunasin is y = 8.975x.

[0064] 2) Optimum pH of Fe3O4-Rha.

[0065] The optimum pH experiment of the enzyme was determined in sodium citrate-citric acid buffer at pH 4.0, 5.0, 5.7, and NaH2PO4-Na2HPO4 buffer at pH 6.0, 7.0, 8.0. The reaction temperature was 40 °C, and the reaction time was 60 min. Then, carry out the reaction according to the method in step (1). Each group had three parallels. Detect and determine the enzyme activities of Rha enzyme and Fe3O4-Rha by HPLC. Take the average value of the group with the highest activity as the highest enzyme activity and define it as 100%, and then calculate the relative enzyme activities of other groups.

[0066] The results are as Figure 18 shown. In the range of pH 4.0 - 8.0, the enzyme activities of the free enzyme and the immobilized enzyme Fe3O4-Rha first increased and then decreased, and both had the maximum activity at pH 6.0, indicating that the immobilized enzyme Fe3O4-Rha maintained the properties of the original enzyme.

[0067] 3) Optimum temperature of Fe3O4-Rha.

[0068] Measurements were carried out at 10°C, 20°C, 30°C, 40°C, 50°C, and 60°C respectively according to the method of step (1). The buffer solution was 50 mM NaH2PO4-Na2HPO4 buffer solution with pH 6.0. The reaction time was 60 min. There were three parallels in each group. The activities of Rha enzyme and Fe3O4-Rha enzyme were detected by HPLC. The average value of the group with the highest activity was taken as the highest enzyme activity and defined as 100%, and the relative enzyme activities of other groups were calculated accordingly.

[0069] The results are as Figure 19 shown. In the range of 10°C to 60°C, the enzyme activities of free enzyme and immobilized enzyme Fe3O4-Rha first increased and then decreased, and both had the maximum activity at 40°C, indicating that the immobilized enzyme Fe3O4-Rha maintained the properties of the original enzyme.

[0070] 4) Organic solvent tolerance of Fe3O4-Rha.

[0071] For the organic solvent tolerance experiment, several organic reagents such as methanol, ethanol, isopropanol, ethylene glycol, and dimethyl sulfoxide (DMSO) were selected. The proportion of organic solvent was 5% (v / v). The reaction was as follows: After mixing 170 µL of 50 mM phosphate buffer solution with pH 6.0, 10 µL of organic solvent or ultrapure water and 10 µL of 20 mM naringin (dissolved in DMSO), incubate at 40°C for 2 min, then add 10 µL of free enzyme and immobilized enzyme Fe3O4-Rha respectively. The reaction time was 60 min, and then the reaction was carried out according to the method of step (1). There were 3 parallels in each group. The activities of Rha enzyme and Fe3O4-Rha enzyme were detected and measured by HPLC. The control group used ultrapure water to replace the corresponding organic solvent and defined its enzyme activity as 100%, and the relative enzyme activities of other groups were calculated accordingly.

[0072] The results are as Figure 20 shown. The organic solvent tolerance of Fe3O4-Rha is better than that of free enzyme. In the reaction system containing ethanol, the relative enzyme activities of free enzyme and immobilized enzyme Fe3O4-Rha are 65.06% and 81.77% respectively. In the reaction system containing isopropanol, the relative enzyme activities of free enzyme and immobilized enzyme Fe3O4-Rha are 51.29% and 74.97% respectively. The immobilized enzyme Fe3O4-Rha is about 23.68% higher than the free enzyme, which is significantly better than the free enzyme. This may be because after the Rha enzyme is covalently bound to Fe3O4 nanoparticles, its structural rigidity is enhanced, resulting in a more stable spatial conformation.

[0073] (4) Biotransformation of naringin by Fe3O4-Rha.

[0074] 1) Effect of enzyme concentration on conversion rate.

[0075] The Fe3O4-Rha reaction buffer was 50 mM phosphate buffer at pH 6.0, the reaction temperature was 40 °C, the reaction time was 60 min, and the final concentrations of the immobilized enzyme Fe3O4-Rha were 0.05 µg / µL, 0.1 µg / µL, 0.15 µg / µL, 0.2 µg / µL, 0.25 µg / µL, 0.3 µg / µL, and 0.35 µg / µL, with 3 parallels in each group. The reaction was carried out according to the method in step (1). After the reaction was terminated, it was centrifuged at 13,000 rpm for 5 min with an ultra-high-speed refrigerated centrifuge, the supernatant was filtered through a 0.22 µm organic filter membrane, detected by HPLC, and the conversion of naringin by Fe3O4-Rha was calculated according to the peak area.

[0076] The results are as Figure 21 shown. As the concentration of the immobilized enzyme Fe3O4-Rha gradually increased, the conversion of naringin gradually increased. When the concentration of the immobilized enzyme Fe3O4-Rha was 0.3 µg / µL, the conversion rate reached 95.71%; when the concentration of the immobilized enzyme Fe3O4-Rha was 0.35 µg / µL, the conversion rate was 96.27%, indicating that naringin was basically completely converted.

[0077] 2) Effect of reaction time on the conversion rate.

[0078] The Fe3O4-Rha reaction buffer was 50 mM phosphate buffer at pH 6.0, the reaction temperature was 40 °C, and the enzymatic reaction times were 10, 20, 30, 40, 50, 60, and 70 min, with 3 parallels in each group. The reaction was carried out according to the method in step (1). After the reaction was terminated, it was centrifuged at 13,000 rpm for 5 min with an ultra-high-speed refrigerated centrifuge, the supernatant was filtered through a 0.22 µm organic filter membrane, detected by HPLC, and the conversion of naringin by Fe3O4-Rha was calculated according to the peak area.

[0079] The results are as Figure 22 shown. As the enzymatic reaction time gradually increased, the conversion of naringin gradually increased. After 70 min of enzymatic reaction, the conversion rate reached 96.33%, indicating that naringin was basically completely converted.

[0080] 3) Effect of substrate concentration on the conversion rate The substrate for the Fe3O4-Rha enzymatic reaction was 10 µL of 40 mM naringin, the reaction buffer was 50 mM phosphate buffer at pH 6.0, the reaction temperature was 40 °C, and the reaction times were 20, 40, 60, 80, 100, and 120 min, with 3 parallels in each group. The reaction was carried out according to the method in step (1). When the reaction was terminated, 100 µL was taken from the 200 µL reaction mixture and added to 900 µL of methanol (chromatographically pure) to terminate the reaction. Then, it was centrifuged at 13,000 rpm for 5 min using an ultra-high-speed refrigerated centrifuge. The supernatant was filtered through a 0.22 µm organic filter membrane and detected by HPLC. The conversion of naringin by Fe3O4-Rha was calculated based on the peak area.

[0081] The results are as Figure 23 shown. With the gradual increase of the enzymatic reaction time, the conversion of naringin gradually increased. When the enzymatic reaction proceeded to 80 min, the conversion rate reached 91.14%. When the enzymatic reaction time increased to 100 min and 120 min, the naringin was completely converted.

[0082] 4) HPLC chromatogram of the biotransformation of naringin by Fe3O4-Rha.

[0083] Naringin and prunasin were respectively prepared into 20 mM stock solutions with DMSO as the solvent, and then diluted to 0.4 mM with chromatographic-grade methanol, and then mixed in a 1:1 (v / v) ratio and diluted with each other to a final concentration of 0.2 mM each, serving as the standards for naringin and prunasin. 10 µL of 50 mM phosphate buffer at pH 8.0 was used to replace 10 µL of the enzyme, and the reaction was carried out according to the method in step (1) for 60 min. The solution filtered through the organic filter membrane after the reaction was used as the control group. 0.3 µg of Rha was added, and the reaction was carried out according to the method in step (1) for 60 min. The solution filtered through the organic filter membrane after the reaction was used as the free enzyme group. 15 µg of immobilized enzyme Fe3O4-Rha was added, and the reaction was carried out according to the method in step (1) for 60 min. The solution filtered through the organic filter membrane after the reaction was used as the immobilized enzyme group. Finally, it was detected on a high-performance liquid chromatography instrument to obtain the corresponding high-performance liquid chromatography diagram.

[0084] The results are as Figure 24 shown, Figure 24 where A is the high-performance liquid chromatography result diagram of the equal-proportion mixture of naringin and prunasin, and chromatographic peaks of naringin and prunasin appeared at 4.33 min and 5.01 min respectively. Figure 24 where B is the high-performance liquid chromatography result diagram of the control group, and only the peak of naringin could be detected at 4.33 min, indicating that naringin was not converted. Figure 24 where C is the high-performance liquid chromatography result diagram of the free enzyme group, Figure 24Figure D shows the HPLC results of the immobilized enzyme group. It can be seen that in the HPLC chromatograms after adding free enzyme and immobilized enzyme Fe3O4-Rha, in addition to the chromatographic peaks of naringin appearing at 4.35 min and 4.33 min respectively, peaks of the product prunin were also detected at 5.04 min and 5.01 min, indicating that both the free enzyme and the immobilized enzyme Fe3O4-Rha can catalyze the conversion of naringin to prunin.

[0085] (5)Reusability of Fe3O4-Rha.

[0086] Mix 180 µL of 50 mM phosphate buffer at pH 6.0 with 10 µL of 20 mM naringin (dissolved in DMSO), incubate at 40 °C for 2 min, with 3 parallels in each group. Add 10 µL of Fe3O4-Rha, shake and react at 800 rpm in a thermostatic mixer for 60 min. After the reaction, immediately perform magnetic separation, transfer 150 µL of the supernatant to 600 µL of methanol (chromatographically pure) to terminate the reaction, then discard the remaining 50 µL of the supernatant in the tube, add 190 µL of 50 mM phosphate buffer at pH 6.0, and then add 10 µL of 20 mM naringin for the next cycle reaction. React in this way for 10 cycles, finally centrifuge and filter with a 0.22 µm organic filter membrane, and finally use HPLC to detect the reaction results. Take the enzyme activity measured in the first reaction result as 100%, and calculate the relative enzyme activity of other groups.

[0087] The results are as Figure 25 shown. After 5 cycles of reaction, the relative activity of Fe3O4-Rha was 61.34%. After 7 cycles of reaction, the relative activity decreased to 31.08%, indicating that the immobilized enzyme Fe3O4-Rha has good reusability.

[0088] (6)Scale-up reaction of Fe3O4-Rha.

[0089] Add 90 mL of phosphate buffer (50 mM at pH 6.0), 5 mL of the substrate naringin (40 mM), and 5 mL of the immobilized enzyme Fe3O4-Rha (1 µg / µL) to a 250 mL flask, mix well, and perform a shaking reaction at 200 rpm and 40 °C. Take 100 µL from the reaction mixture at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 14 h at the start, take 3 times, and then add 900 µL of methanol (chromatographically pure) to terminate the reaction. After the reaction is terminated, centrifuge at 13000 rpm for 5 min with an ultra-speed refrigerated centrifuge, filter the supernatant with a 0.22 µm organic filter membrane, and finally use HPLC to detect the conversion of naringin.

[0090] The results are as Figure 26As shown, after 8 h of reaction, the conversion rate reached 91.5%. When the reaction time was further extended to 10 h, 12 h, and 14 h, the conversion rates were 94.06%, 95.57%, and 98.01% respectively, with a slight increase in the conversion rate, indicating that the reaction was basically completed. The above results show that after scaling up the reaction system, the immobilized enzyme Fe3O4-Rha can still well catalyze the conversion of naringin.

[0091] Therefore, the Fe3O4 nanoparticles provided by the present invention have good superparamagnetism; the immobilized enzyme Fe3O4-Rha prepared with this as the carrier has good magnetic responsiveness and superparamagnetism, with an optimal pH of 6.0, an optimal temperature of 40 °C, and better tolerance to organic solvents than free enzymes; it can be used for the efficient biotransformation of naringin, with a conversion rate of up to 98.01%; it has good reusability, and after being recycled 5 times, the relative enzyme activity is 61.34%; after scaling up the system, the immobilized enzyme Fe3O4-Rha can still well catalyze the conversion of naringin; the provided preparation method is simple and easy to operate, the prepared Fe3O4 nanoparticles have good morphology and water dispersibility, and the immobilization efficiency of α-L-rhamnosidase is 49.40%; the requirements for equipment are low, no toxic reagents are used, and no toxic and harmful substances are generated, which is green and environmentally friendly.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A preparation method of immobilized enzyme Fe3O4-Rha, characterized in that, It includes the following steps: S1. Prepare hydrophobic Fe3O4 nanoparticles. Stir and mix iron acetylacetonate, 1,2-dodecanediol, oleic acid, oleylamine, and benzyl ether under a protective atmosphere, and then react in an air atmosphere to obtain a black mixture. After cooling to room temperature, add anhydrous ethanol and centrifuge. Add oleic acid, oleylamine, and n-hexane to the precipitate in sequence, mix well and centrifuge to take the supernatant, then add anhydrous ethanol and centrifuge to take the precipitate, which is the hydrophobic Fe3O4 nanoparticles; S2. Perform phase transfer on the hydrophobic Fe3O4 nanoparticles prepared in S1. Disperse the hydrophobic Fe3O4 nanoparticles in tetrahydrofuran to obtain a Fe3O4 nanoparticle dispersion; add tetrahydrofuran to 3,4-dihydroxycinnamic acid, dissolve it and then dropwise add the prepared Fe3O4 nanoparticle dispersion, and react under a protective atmosphere. After the reaction ends, cool to room temperature, add NaOH solution and centrifuge, and the precipitate is the hydrophilic Fe3O4 nanoparticles; S3. Activate the hydrophilic Fe3O4 nanoparticles prepared in S2. Disperse the hydrophilic Fe3O4 nanoparticles prepared in S2 in ultrapure water, add a crosslinking agent and PBS buffer solution in sequence, oscillate and incubate, then perform magnetic separation, and remove the crosslinking agent to obtain the activated Fe3O4 nanoparticles; S4. Disperse the activated Fe3O4 nanoparticles obtained in S3 in PBS buffer solution to obtain an activated Fe3O4 nanoparticle PBS dispersion. Add α-L-rhamnosidase Rha and carry out a curing reaction on a shaker. After the reaction ends, perform magnetic separation to obtain the immobilized enzyme Fe3O4-Rha.

2. The preparation method of the immobilized enzyme Fe3O4-Rha according to claim 1, wherein: In S1, the molar ratio of iron acetylacetonate: 1,2-dodecanediol: oleic acid: oleylamine: benzyl ether is 1:5:3:3:53; the stirring and mixing condition is to stir and react at 200 °C for 2 h; the reaction condition in the air atmosphere is 300 °C and the reaction is for 1 h.

3. The preparation method of an immobilized enzyme Fe3O4-Rha according to claim 1, characterized in that: In S1, the volume ratio of the added black mixture to anhydrous ethanol is 1:2 - 3; the volume ratio of the supernatant to anhydrous ethanol is 1:2 - 3; the volume ratio of oleic acid: oleylamine: n-hexane is 1:1:

200.

4. The preparation method of an immobilized enzyme Fe3O4-Rha according to claim 1, characterized in that: In S2, the mass-volume ratio of hydrophobic Fe3O4 nanoparticles: tetrahydrofuran: 3,4-dihydroxycinnamic acid is 20 mg: 11 mL: 50 mg; 3,4-dihydroxycinnamic acid is dissolved in tetrahydrofuran under a protective atmosphere; the reaction condition under the protective atmosphere is to react at 50 °C for 3 h.

5. The preparation method of an immobilized enzyme Fe3O4-Rha according to claim 1, characterized in that: In S2, the concentration of the NaOH solution is 0.5 M; the volume ratio of the addition amount of the NaOH solution to the volume of the nanoparticle mixed solution is 1:

22.

6. The preparation method of an immobilized enzyme Fe3O4-Rha according to claim 1, characterized in that: In S3, the crosslinking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1.2:1; when activating the Fe3O4 nanoparticles, the mass-volume ratio of hydrophilic Fe3O4 nanoparticles: ultrapure water: crosslinking agent: PBS buffer solution is 10 mg: 1 mL: 4.4 mL: 4.4 mL.

7. The preparation method of an immobilized enzyme Fe3O4-Rha according to claim 1, characterized in that: In S3, the conditions for oscillating and incubating are at room temperature, 150 - 200 rpm, and oscillate and incubate for 20 - 40 min; to remove the crosslinking agent, wash with PBS buffer solution at pH 7.

0.

8. The preparation method of an immobilized enzyme Fe3O4-Rha according to claim 1, characterized in that: The mass ratio of the activated Fe3O4 nanoparticles to α-L-rhamnosidase Rha in S4 is 1:2; the curing reaction conditions are 4 °C, 150 - 200 rpm, and 2 - 5 h.

9. An immobilized enzyme Fe3O4-Rha prepared by the method for preparing an immobilized enzyme Fe3O4-Rha according to any one of claims 1 - 8.

10. Use of the immobilized enzyme Fe3O4-Rha as described in claim 9 in the preparation of a new compound, characterized in that: The new compound is achieved by cleaving the α-L-rhamnoside bond.

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