Preparation and application of glucose oxidase / acyltransferase cross-linked enzyme aggregate

Abstract

The invention provides preparation and application of a glucose oxidase / acyltransferase cross-linked enzyme aggregate. The preparation method comprises the following steps: firstly preparing Fe3O4 (at) SiO2 magnetic nanoparticles, modifying the surfaces of the magnetic nanoparticles with amino groups, and then immobilizing glucose oxidase and acyltransferase on the amino-functionalized Fe3O4 (at) SiO2 magnetic nanoparticles by a glutaraldehyde crosslinking method to obtain the glucose oxidase / acyltransferase crosslinked enzyme aggregate. The GOD enzyme activity recovery rate and the MsAcT enzyme activity recovery rate of the glucose oxidase / acyltransferase cross-linked enzyme aggregate can reach 85.6% and 98.2% respectively, the cascade efficiency is excellent, and the catalytic efficiency is 2.4 times of the cascade efficiency of free enzyme catalysis. The glucose oxidase / acyltransferase cross-linked enzyme aggregate can be easily separated from a reaction system, and has good reaction selectivity in catalysis of a hydrolysis reaction and an esterification reaction.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biotechnology, green chemical engineering, and biomanufacturing technology, and particularly relates to the preparation and application of a glucose oxidase / acyltransferase cross-linked enzyme aggregate. Background Art

[0002] To establish a multi-enzyme cascade catalytic reaction system to achieve the "one-pot" catalysis of multi-step cascade chemical reactions by multiple enzymes, where the intermediate products do not need to be separated and purified and are directly converted into the end products, which can not only improve the conversion rate, but also effectively reduce the production cost, especially suitable for various cascade chemical reactions with unstable intermediate products.

[0003] Different enzyme proteins have their own physical and chemical properties and optimal catalytic conditions. In the process of catalyzing cascade reactions by combining different enzyme proteins, there are still quite challenges in effectively achieving the maximum catalytic efficiency. There are methods such as using DNA scaffold technology to regulate the positioning and spacing of enzyme proteins in space to optimize the catalytic efficiency of the cascade system; or using immobilization technology to create a microenvironment (pH environment) suitable for each enzyme to optimize the catalytic efficiency of the cascade system; or using protein engineering technology to modify some enzymatic properties of a certain enzyme in order to improve the catalytic efficiency of cascade catalysis. The above methods all have cumbersome operation processes, high technical difficulties, and high costs. Most of the technologies are still only in the laboratory research and development stage, and there are still many problems to be overcome in large-scale preparation.

[0004] Multi-enzyme cross-linked aggregates precipitate a mixture of two or more kinds of enzymes using a precipitant; then glutaraldehyde is used to directly cross-link and aggregate different enzyme proteins together to form a co-immobilized enzyme preparation in the size of nanoparticles. The above preparation method is simple in operation and low in cost, and is suitable for large-scale industrial preparation. In most cases, the multi-enzyme cross-linked enzyme preparations prepared by the above strategy can all show good catalytic effects. However, when preparing multi-enzyme cross-linked enzyme aggregates of glucose oxidase (Glucose oxidase, GOD) and other enzyme proteins (such as Candida antarctica lipase B (CALB), porcine liver esterase, horseradish peroxidase, etc.) using the above method, since the surface of glucose oxidase is rich in lysine residues (each glucose oxidase molecule contains 15 lysine residues on its surface), there is a significant difference in the content of lysine residues on the surface of most enzyme proteins (for example, there are only 4 lysines on the surface of a single subunit of Mycobacterium smegmatis acyltransferase MsAcT). Therefore, it is very difficult to select a suitable concentration of glutaraldehyde (the conventional dosage of glutaraldehyde concentration is likely to cause over-cross-linking of glucose oxidase; while too low a glutaraldehyde concentration will lead to low cross-linking degree of Mycobacterium smegmatis acyltransferase MsAcT cascaded with glucose oxidase and low enzyme activity recovery rate). To solve this problem, the currently adopted strategies basically all use other cross-linking agents, such as cross-linking agents like Genipin, etc. However, the high price of the above cross-linking agents makes the prepared multi-enzyme cross-linked enzyme preparations expensive and difficult to promote and popularize.

[0005] Therefore, it is of great significance to develop a new preparation method for glucose oxidase / acyltransferase cross-linked enzyme aggregates. Summary of the Invention

[0006] In order to overcome the deficiencies of the prior art, the present invention provides a preparation and application of a glucose oxidase / acyltransferase cross-linked enzyme aggregate.

[0007] To achieve the above object. The technical scheme adopted by the present invention is as follows: The first aspect of the present invention provides a preparation method for a glucose oxidase / acyltransferase cross-linked enzyme aggregate, comprising the following steps: 1) Prepare Fe3O4@SiO2 magnetic nanoparticles; 2) Prepare amino-functionalized Fe3O4@SiO2 magnetic nanoparticles; 3) Fix glucose oxidase and acyltransferase on amino-functionalized Fe3O4@SiO2 magnetic nanoparticles by the method of cross-linking with glutaraldehyde to obtain a glucose oxidase / acyltransferase cross-linked enzyme aggregate.

[0008] The specific steps of step 1) are as follows: 0.8 g of nano-Fe3O4 is ultrasonically dispersed in a mixed solution composed of 36 mL of absolute ethanol and 4 mL of single-distilled water. 1.6 mL of tetraethyl orthosilicate is added, and then PEG1000 with a final concentration of 1 mg / mL is added. Then, 10 mL of 25 wt% ammonia water is added under stirring, and the reaction is stirred at room temperature for 12 h. Then, centrifugation is carried out to collect the precipitate, which is washed with single-distilled water and absolute ethanol respectively, and then dried overnight at 60 °C to obtain Fe3O4@SiO2 magnetic nanoparticles.

[0009] The specific steps of step 2) are as follows: 0.25 g of Fe3O4@SiO2 magnetic nanoparticles is ultrasonically dispersed in 20 mL of absolute ethanol, and PEG1000 with a final concentration of 1 mg / mL is added. The pH is adjusted to 4 - 8, and then 0.5 mL of 3-aminopropyltriethoxysilane is added. The reaction is carried out on a shaker at 20 - 40 °C for 12 h. Then, centrifugation is carried out to collect the precipitate, which is washed with single-distilled water and absolute ethanol respectively, and then dried overnight at 60 °C to obtain amino-functionalized Fe3O4@SiO2 magnetic nanoparticles.

[0010] Preferably, the specific steps of step 2) are as follows: 0.25 g of Fe3O4@SiO2 magnetic nanoparticles is ultrasonically dispersed in 20 mL of absolute ethanol, and PEG1000 with a final concentration of 1 mg / mL is added. The pH is adjusted to 4, and then 0.5 mL of 3-aminopropyltriethoxysilane is added. The reaction is carried out on a shaker at 40 °C for 12 h. Then, centrifugation is carried out to collect the precipitate, which is washed with single-distilled water and absolute ethanol respectively, and then dried overnight at 60 °C to obtain amino-functionalized Fe3O4@SiO2 magnetic nanoparticles.

[0011] The specific steps of step 3) are as follows: 100 mg of amino-functionalized Fe3O4@SiO2 magnetic nanoparticles is ultrasonically dispersed in 1 mL of sodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution with pH 7.0 and 50 mmol / L. Glutaraldehyde with a final concentration of 5 - 80 mmol / L is added, and the reaction is carried out at 25 °C and 220 rpm for 1.5 h. After magnetic separation and washing with sodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution with pH 7.0 and 50 mmol / L, it is resuspended in 900 μL of sodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution with pH 7.0 and 50 mmol / L to obtain a resuspension; 100 μL of GOD / MsAcT mixed aqueous solution with a total protein content of 10 mg / mL is added to the obtained resuspension, and the reaction is carried out at 25 °C and 150 rpm for 1.5 h. After magnetic separation and washing with sodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution with pH 7.0 and 50 mmol / L, glucose oxidase / acyltransferase cross-linked enzyme aggregates are obtained.

[0012] Preferably, step 3) is specifically as follows: Ultrasonically disperse 100 mg of amino-functionalized Fe3O4@SiO2 magnetic nanoparticles in 1 mL of a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 and a concentration of 50 mmol / L. Add glutaraldehyde with a final concentration of 20 mmol / L and react at 25 °C and 220 rpm for 1.5 h. After magnetic separation and washing with a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 and a concentration of 50 mmol / L, resuspend in 900 μL of a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 and a concentration of 50 mmol / L to obtain a resuspension. Add 100 μL of a GOD / MsAcT mixed aqueous solution with a total protein content of 10 mg / mL to the obtained resuspension and react at 25 °C and 150 rpm for 1.5 h. After magnetic separation and washing with a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 and a concentration of 50 mmol / L, a glucose oxidase / acyltransferase cross-linked enzyme aggregate is obtained.

[0013] The enzyme activity ratio of GOD:MsAcT in the above GOD / MsAcT mixed aqueous solution is 50:1.

[0014] The second aspect of the present invention provides a glucose oxidase / acyltransferase cross-linked enzyme aggregate, which is prepared by the above preparation method.

[0015] The third aspect of the present invention provides the application of the above glucose oxidase / acyltransferase cross-linked enzyme aggregate in the catalytic oxidation reaction of enols.

[0016] The above enols include citronellol, 6-methyl-5-hepten-2-ol, and dihydrolinalool.

[0017] The remarkable advantages of the present invention are as follows: The present invention first prepares Fe3O4@SiO2 magnetic nanoparticles, then amino-functionalizes the Fe3O4@SiO2 magnetic nanoparticles, and then immobilizes glucose oxidase and acyltransferase on the amino-functionalized Fe3O4@SiO2 magnetic nanoparticles by glutaraldehyde cross-linking to obtain a glucose oxidase / acyltransferase cross-linked enzyme aggregate. The glucose oxidase / acyltransferase cross-linked enzyme aggregate prepared by this method is easy to separate from the reaction system. In the catalytic hydrolysis reaction and esterification reaction, it has good reaction selectivity, and the cascade efficiency is excellent. Compared with the glucose oxidase / acyltransferase cross-linked enzyme aggregate prepared by the method of adding a protein protectant and the method of modifying lysine residues on the surface of glucose oxidase to reduce its abundance, as well as the free enzyme catalytic system, the cascade efficiency is higher. Description of the Drawings

[0018] Figure 1 : Principle of PEG-modified protein.

[0019] Figure 2 : SDS-PAGE gel electrophoresis of GOD / MsAcT cross-linked enzyme aggregates at different modifier concentrations. M: Marker; 1: Unmodified; 2: Final modifier concentration of 40 mmol / L; 3: Final modifier concentration of 80 mmol / L; 4: Final modifier concentration of 120 mmol / L.

[0020] Figure 3 : Flow chart of co-immobilization of GOD and MsAcT on amino-functionalized Fe3O4@SiO2 magnetic nanoparticles.

[0021] Figure 4 : Product chromatogram of the oxidation reaction of citronellol catalyzed by GOD / MsAcT cross-linked enzyme aggregates. 1: Citronellol (18.924 min); 4: Internal standard (21.639 min); 5: Citronellyl acetate (26.760 min); 7: Hydroxycitronellal (37.228 min); 8: cis-4-Methylcyclohexanol (43.867 min); 9: trans-4-Methylcyclohexanol (44.060 min).

[0022] Figure 5 : Product chromatogram of the oxidation reaction of 6-methyl-5-hepten-2-ol catalyzed by GOD / MsAcT cross-linked enzyme aggregates. 4: Internal standard (14.672 min); 15: (±)-6-Methyl-5-hepten-2-ol (5.706 min); 18: Transesterification product of 6-methyl-5-hepten-2-ol (8.503 min); 13 (5.300 min), 14 (5.493 min), 16 (6.411 min), 17 (6.696 min): Epoxides of 6-methyl-5-hepten-2-ol; 19: Other products (19.483 min).

[0023] Figure 6 : Product chromatogram of the oxidation reaction of dihydrolinalool catalyzed by GOD / MsAcT cross-linked enzyme aggregates. 4: Internal standard (14.672 min); 20 (8.424 min), 21 (8.552 min), 23 (11.454 min), 24 (11.567 min): Epoxides of dihydrolinalool; 22: Dihydrolinalool (9.537 min); 25 (19.666 min), 26 (19.900 min): Other products. Detailed implementation manners

[0024] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0025] The glucose oxidase (GOD) used in the following examples is a commercially available product of Sangon Biotech (Shanghai) Co., Ltd., CAS: 9001-37-0, EINECS number: 232-601-0; the preparation method of Mycobacterium smegmatis acyltransferase (MsAcT) has been disclosed in Patent CN202211116546.4, specifically MsAcT-Ser in Patent CN202211116546.4 54 Ile / Leu 119 Gln.

[0026] In the following examples, for the method of measuring the enzyme activity of the cascade system, the total volume of the reaction system is 200 μL, including: 143 μL of sodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH 8.0 and 50 mmol / L, 20 μL of glucose with a final concentration of 50 mmol / L, 15 μL of triacetin with a final concentration of 15 mmol / L, 15 μL of NaBr with a final concentration of 150 mmol / L, 2 μL of 2-chloro-5,5-diethyl-1,3-cyclohexanedione (Monochlorodimedone, MCD) with a final concentration of 0.05 mmol / L, and 5 μL of GOD / MsAcT cross-linked enzyme aggregates. The reaction system is reacted at 37 °C for 5 min, and the change value of MCD is recorded at 290 nm.

[0027] Example 1: Preparation of GOD / MsAcT cross-linked enzyme aggregates by the strategy of adding protectants 1.1 Screening of precipitants Add 900 μL of precipitant to 100 μL of GOD aqueous solution with a protein content of 10 mg / mL and 100 μL of MsAcT aqueous solution with a protein content of 10 mg / mL respectively, let it stand and precipitate at 4 °C for 2 h, then add glutaraldehyde with a final concentration of 10 mmol / L to the system, cross-link at 25 °C and 150 rpm for 1.5 h, then centrifuge at 8000 rpm for 5 min, collect the precipitate, wash it with sodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH 8.0 and 50 mmol / L and resuspend it, measure the GOD enzyme activity, MsAcT enzyme activity and protein content of the resuspended solution, and calculate the enzyme activity recovery rate and protein recovery rate. The precipitant is selected from any one of the following: ammonium sulfate with 90% saturation (0 °C), ethylene glycol dimethyl ether, polyethylene glycol 800, acetone, isopropanol, tert-butanol.

[0028] The results are shown in Table 1. When isopropanol was used as the precipitant, the enzyme activity recovery rates of GOD and MsAcT were 31.0% and 60.9% respectively; when ethylene glycol dimethyl ether was used as the precipitant, the enzyme activity recovery rate of MsAcT reached 68.4%, but at this time the enzyme activity recovery rate of GOD was only 2.2%. Therefore, isopropanol was selected as the precipitant for the subsequent preparation of glucose oxidase / acyltransferase cross-linked enzyme aggregates.

[0029] Table 1 Effects of precipitants on the enzyme activities of GOD and MsAcT Note: The lowercase letters in the table represent the results of significance analysis. Two groups with the same letter in the same column do not have significant differences (P < 0.05).

[0030] 1.2 Effects of cross-linker concentration on the enzyme activities of cross-linked enzyme aggregates ① Respectively, 900 μL of isopropanol was added to 100 μL of GOD aqueous solution with a protein content of 10 mg / mL and 100 μL of MsAcT aqueous solution with a protein content of 10 mg / mL. The mixture was allowed to stand at 4 °C for 2 h for precipitation, and then glutaraldehyde with a final concentration of 5 - 100 mmol / L was added to the system. The cross-linking reaction was carried out at 25 °C and 150 rpm for 1.5 h. Then, the mixture was centrifuged at 8000 rpm for 5 min to collect the precipitate. The precipitate was washed with a sodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution with pH 8.0 and 20 mmol / L and then resuspended. The GOD enzyme activity, MsAcT enzyme activity and protein content of the resuspended solution were measured, and the enzyme activity recovery rate and protein recovery rate were calculated.

[0031] ② 900 μL of isopropanol was added to 100 μL of GOD / MsAcT mixed aqueous solution with a total protein content of 10 mg / mL (the enzyme activity ratio of GOD:MsAcT was 50:1). The mixture was allowed to stand at 4 °C for 2 h for precipitation, and then glutaraldehyde with a final concentration of 5 - 100 mmol / L was added to the system. The cross-linking reaction was carried out at 25 °C and 150 rpm for 1.5 h. Then, the mixture was centrifuged at 8000 rpm for 5 min to collect the precipitate. The precipitate was washed with a sodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution with pH 8.0 and 20 mmol / L and then resuspended. The GOD enzyme activity, MsAcT enzyme activity, total protein content of the resuspended solution and the catalytic efficiency of the cascade system were measured, and the enzyme activity recovery rate and total protein recovery rate were calculated.

[0032] The results are shown in Table 2. The protein recovery rate increased with the increase of the final concentration of the cross-linker glutaraldehyde; when the final concentration of glutaraldehyde was 10 mmol / L, the GOD enzyme activity recovery rate and MsAcT enzyme activity recovery rate of the GOD / MsAcT cross-linked enzyme aggregates were the highest, showing significant differences compared with other groups. And at this time, the degradation rate of MCD under the same MsAcT enzyme activity unit was the highest, indicating that the catalytic efficiency of the cascade system under this cross-linking condition was the highest, showing a significant improvement compared with the free double-enzyme cascade system.

[0033] Table 2 Effect of Glutaraldehyde Concentration on Enzyme Activity of Crosslinked Enzyme Aggregates Table 2 (continued) Effect of Glutaraldehyde Concentration on Enzyme Activity of Crosslinked Enzyme Aggregates Note: Lowercase letters in the table represent the results of significance analysis. Two groups with the same letter in the same column do not have significant differences (P < 0.05).

[0034] 1.3 Effect of Adding Protective Agent BSA on the Cascade Efficiency of GOD / MsAcT Crosslinked Enzyme Aggregates in the Immobilization System To 100 μL of a mixed aqueous solution of GOD / MsAcT with a total protein content of 10 mg / mL (the enzyme activity ratio of GOD:MsAcT is 50:1), 0 - 1 mg of BSA was added, then 900 μL of isopropanol was added, and the mixture was allowed to stand and precipitate at 4°C for 2 h. Then, glutaraldehyde with a final concentration of 10 mmol / L was added to the system, and crosslinking was carried out at 25°C and 150 rpm for 1.5 h. Then, centrifugation was performed at 8000 rpm for 5 min, and the precipitate was collected, washed with a sodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution at pH 8.0 and 20 mmol / L, and resuspended. The GOD enzyme activity, MsAcT enzyme activity, total protein content of the resuspended solution, and catalytic efficiency of the cascade system were measured, and the enzyme activity recovery rate and total protein recovery rate were calculated.

[0035] The results are shown in Table 3. When the addition amount of BSA is 0.1 mg, the GOD enzyme activity recovery rate and MsAcT enzyme activity recovery rate of the GOD / MsAcT crosslinked enzyme aggregates are the highest, which are 47.0% and 85.6% respectively, showing a significant increase compared with the case without adding BSA. At this time, although the degradation rate of MCD under the same MsAcT enzyme activity unit has no significant difference compared with the cases when the addition amount of BSA is 0 - 0.05 mg and 0.2 - 1 mg, compared with other groups, the enzyme activity recovery rate of the single enzyme is significantly increased.

[0036] Table 3 Effect of BSA on Enzyme Activity of Crosslinked Enzyme Aggregates Note: Lowercase letters in the table represent the results of significance analysis. Two groups with the same letter in the same column do not have significant differences (P < 0.05).

[0037] Example 2: Preparation of GOD / MsAcT Crosslinked Enzyme Aggregates by PEG Modification of GOD 2.1 PEG Modification of GOD The process of PEG modification of GOD is as Figure 1As shown in the figure. The reaction system is specifically shown in Table 4. The buffer solution therein is a pH 6.0, 50 mmol / L Na2HPO4-NaH2PO4 buffer solution containing 50 mmol / L KCl, and the modifier is polyethylene glycol dicarboxylic acid (average molecular weight 600); the final concentrations of the modifier in reaction system 1, reaction system 2, and reaction system 3 are 40 mmol / L, 80 mmol / L, and 120 mmol / L, respectively. React the reaction system in a water bath at 25 °C for 2 h. After the reaction is completed, wash it 3 times with an ultrafiltration tube to obtain GOD-PEG, and then measure its enzyme activity and perform SDS-PAGE electrophoresis detection respectively.

[0038] Table 4 Composition of the reaction system The results of the effect of the final concentration of the modifier on the GOD enzyme activity are shown in Table 5. As the final concentration of the modifier increases, the residual enzyme activity of GOD continuously decreases. When the final concentration of the modifier ≤ 80 mmol / L, the residual enzyme activity of GOD reaches more than 80%. The electrophoresis results of GOD modified by PEG are as Figure 2 shown.

[0039] Table 5 Effect of the final concentration of the modifier on the GOD enzyme activity Note: abc in the table represents the results of significance analysis. Two groups with the same letter in the same column do not have significant differences (P < 0.05).

[0040] 2.2 Cascade efficiency of GOD-PEG / MsAcT cross-linked enzyme aggregates Add 900 μL of isopropanol to 100 μL of a mixed aqueous solution of GOD-PEG / MsAcT with a total protein content of 10 mg / mL (the enzyme activity ratio of GOD:MsAcT is 50:1), let it stand and precipitate at 4 °C for 2 h, then add glutaraldehyde with a final concentration of 10 mmol / L to the system, cross-link at 25 °C and 150 rpm for 1.5 h, centrifuge at 8000 rpm for 5 min, collect the precipitate, wash it with a sodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH 8.0 and 50 mmol / L, and resuspend it. Measure the GOD enzyme activity, MsAcT enzyme activity, total protein content, and catalytic efficiency of the cascade system of the resuspended solution, and calculate the enzyme activity recovery rate and total protein recovery rate.

[0041] The results are shown in Table 6. When the final concentration of the modifier is 120 mmol / L, the GOD enzyme activity recovery rate of the GOD-PEG / MsAcT cross-linked enzyme aggregate is 55.5%, and the MsAcT enzyme activity recovery rate is 55.7%.

[0042] Table 6 Effect of the final concentration of the modifier on the enzyme activity of GOD-PEG / MsAcT cross-linked enzyme aggregates Note: abc in the table represents the results of significance analysis. Two groups with the same letter in the same column do not have significant differences (P < 0.05).

[0043] Example 3: Preparation of GOD / MsAcT cross-linked enzyme aggregates by glutaraldehyde cross-linking method 3.1 Preparation of Fe3O4@SiO2 magnetic nanoparticles (Fe3O4@SiO2 MNPs) Disperse 0.8 g of commercially available nano-Fe3O4 (particle size 20 nm) ultrasonically in a mixed solution composed of 36 mL of absolute ethanol and 4 mL of single-distilled water. Add 1.6 mL of tetraethyl orthosilicate (TEOS), then add PEG1000 with a final concentration of 1 mg / mL. Then add 10 mL of 25 wt% ammonia water under stirring at 300 rpm. React at room temperature and 300 rpm for 12 h, then centrifuge at 1200 rpm for 5 min. Collect the precipitate, wash it three times with single-distilled water and absolute ethanol respectively, and then dry it overnight at 60 °C to obtain Fe3O4@SiO2 MNPs.

[0044] 3.2 Influence of pH and temperature on the modification of 3-aminopropyltriethoxysilane (APTES) Disperse 0.25 g of Fe3O4@SiO2 MNPs ultrasonically in 20 mL of absolute ethanol, add PEG1000 with a final concentration of 1 mg / mL, adjust the pH to 4 - 8 with glacial acetic acid, then add 0.5 mL of APTES, react at 20 °C and 300 rpm for 12 h, then centrifuge at 12000 rpm for 5 min. Collect the precipitate, wash it three times with single-distilled water and absolute ethanol respectively, and then dry it overnight at 60 °C to obtain amino-functionalized Fe3O4@SiO2 MNPs. Using glycine as the standard solution, the content of free amino groups in amino-functionalized Fe3O4@SiO2 MNPs was determined by the o-phthalaldehyde method.

[0045] Disperse 0.25 g of Fe3O4@SiO2 MNPs ultrasonically in 20 mL of absolute ethanol, add PEG1000 with a final concentration of 1 mg / mL, adjust the pH to 4 with glacial acetic acid, then add 0.5 mL of APTES, react at 20 - 60 °C and 300 rpm for 12 h, then centrifuge at 12000 rpm for 5 min. Collect the precipitate, wash it three times with single-distilled water and absolute ethanol respectively, and then dry it overnight at 60 °C to obtain amino-functionalized Fe3O4@SiO2 MNPs. Using glycine as the standard solution, the content of free amino groups in amino-functionalized Fe3O4@SiO2 MNPs was determined by the o-phthalaldehyde method.

[0046] The results of the effects of pH and temperature on APTES modification are shown in Tables 7 and 8. When the pH is 4.0 and the reaction temperature is 40 °C, the content of free amino groups in the aminated Fe3O4@SiO2 MNPs is the highest, reaching 458 μmol / g.

[0047] Table 7 Effects of pH on APTES modification Table 8 Effects of temperature on APTES modification 3.3 Preparation of GOD / MsAcT cross-linked enzyme aggregates S1: Ultrasonically disperse 0.25 g of Fe3O4@SiO2 MNPs in 20 mL of absolute ethanol, add PEG1000 with a final concentration of 1 mg / mL, adjust the pH to 4 with glacial acetic acid, then add 0.5 mL of APTES, react on a shaker at 40 °C and 300 rpm for 12 h, then centrifuge at 12000 rpm for 5 min, collect the precipitate, wash the collected precipitate three times with distilled water and absolute ethanol respectively, and then dry overnight at 60 °C to obtain amino-functionalized Fe3O4@SiO2 MNPs.

[0048] S2: Ultrasonically disperse 100 mg of amino-functionalized Fe3O4@SiO2 MNPs in 1 mL of sodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 and a concentration of 50 mmol / L, add glutaraldehyde with a final concentration of 5 - 80 mmol / L, after reacting at 25 °C and 220 rpm for 1.5 h, first adsorb and separate the immobilized enzyme preparation with a magnet, then wash the separated immobilized enzyme preparation three times with sodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 and a concentration of 50 mmol / L, and then resuspend it in 900 μL of sodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 and a concentration of 50 mmol / L to obtain a resuspension; add 100 μL of a mixed aqueous solution of GOD / MsAcT with a total protein content of 10 mg / mL (the enzyme activity ratio of GOD:MsAcT is 50:1) to the obtained resuspension, after reacting at 25 °C and 150 rpm for 1.5 h, first adsorb and separate the immobilized enzyme preparation with a magnet, then wash the separated immobilized enzyme preparation three times with sodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 and a concentration of 50 mmol / L to obtain GOD-MsAcT magnetic Fe3O4 nanoparticle enzyme, that is, GOD / MsAcT cross-linked enzyme aggregates, and resuspend it in sodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 8.0 and a concentration of 50 mmol / L to measure its enzyme activity recovery rate.

[0049] The effects of the final concentration of glutaraldehyde on the enzymatic activity of GOD-MsAcT magnetic Fe3O4 nanoparticles are shown in Table 9. With the increase of the final concentration of glutaraldehyde, the recovery rates of GOD enzymatic activity and MsAcT enzymatic activity of GOD-MsAcT magnetic Fe3O4 nanoparticles both increase first and then decrease. When the final concentration of glutaraldehyde is 20 mmol / L, the recovery rates of GOD enzymatic activity and MsAcT enzymatic activity of GOD-MsAcT magnetic Fe3O4 nanoparticles reach the highest, which are 85.6% and 98.2% respectively. At this time, the enzymatic activity ratio of GOD:MsAcT is 55:1, and the MCD degradation rate per unit enzymatic activity is 6.1×10 -2 μmol / U·min; while the MCD degradation rate per unit enzymatic activity of free GOD and MsAcT at 50:1 is 2.5×10 -2 μmol / U·min, that is, the catalytic efficiency of the GOD-MsAcT magnetic Fe3O4 nanoparticles enzyme in this application is 2.4 times that of the cascade reaction of free GOD and MsAcT.

[0050] Table 9 Effects of the final concentration of glutaraldehyde on the enzymatic activity of GOD / MsAcT cross-linked enzyme aggregates Note: abc in the table represents the results of significance analysis. Two groups with the same letter in the same column do not have significant differences (P<0.05).

[0051] Example 4: Application of GOD / MsAcT cross-linked enzyme aggregates in catalytic reactions S1: Ultrasonically disperse 0.25 g of Fe3O4@SiO2 MNPs in 20 mL of absolute ethanol, add PEG1000 with a final concentration of 1 mg / mL, adjust the pH to 4 with glacial acetic acid, then add 0.5 mL of APTES, react on a shaker at 40 °C and 300 rpm for 12 h, then centrifuge at 12000 rpm for 5 min, collect the precipitate, wash the collected precipitate 3 times with distilled water and absolute ethanol respectively, and then dry overnight at 60 °C to obtain amino-functionalized Fe3O4@SiO2 MNPs.

[0052] S2: 100 mg of amino-functionalized Fe3O4@SiO2MNPs were ultrasonically dispersed in 1 mL of pH 7.0, 50 mmol / L sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution, and glutaraldehyde was added at a final concentration of 20 mmol / L. After reacting at 25 °C and 220 rpm for 1.5 h, the immobilized enzyme preparation was first separated by magnetic adsorption, and then the separated immobilized enzyme preparation was washed three times with pH 7.0, 50 mmol / L sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution, and then resuspended in 900 μL A heavy suspension was obtained by adding 100 μL of a GOD / MsAcT mixed aqueous solution with a total protein content of 10 mg / mL (the enzyme activity ratio of GOD:MsAcT was 50:1) to the obtained heavy suspension, and the reaction was carried out at 25°C and 150 rpm for 1.5 hours. The immobilized enzyme preparation was first separated by magnetic adsorption, and then the separated immobilized enzyme preparation was washed three times with a pH 7.0, 50 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to obtain GOD-MsAcT magnetic Fe3O4 nanoparticle enzyme, i.e., GOD / MsAcT cross-linked enzyme aggregate.

[0053] S3: Oxidation reaction of citronellol: The total volume of the reaction system was 1 mL, including: 114.3 mg GOD / MsAcT cross-linked enzyme aggregate, triacetin with a final concentration of 15 mmol / L, glucose with a final concentration of 50 mmol / L, citronellol with a final concentration of 15 mmol / L, and the rest was disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (50 mmol / L, pH 8.0). The reaction system was reacted at 37°C for 24 h. After the reaction, 20 μL of 250 mmol / L n-decanol was added as an internal standard, and then extracted with 2000 μL n-butanol, and 500 μL of the extract was taken for gas phase detection. The results are shown in Table 10.

[0054] The gas phase detection conditions of citronellol are as follows: the gas chromatography column is HP-5 (30m, 0.32mm, 0.25μm), 60℃ for 1min, 2℃ / min to 150℃. The injection volume is 1μL each time, the carrier gas is nitrogen, the flow rate is 1mL / min, and the split ratio is 50:1. The injection port temperature is 250℃, and the FID temperature is 260℃.

[0055] Total conversion rate C (%) = total mole number of products × 100 / initial mole number of substrates; yield Y (%) = mole number of product A × 100 / initial mole number of substrates; selectivity S (%) = mole number of product A × 100 / total mole number of products.

[0056] S4: Oxidation reaction of 6-methyl-5-hepten-2-ol: The total volume of the reaction system was 1 mL, including: 114.3 mg of GOD / MsAcT cross-linked enzyme aggregates, glycerol triacetate with a final concentration of 15 mmol / L, glucose with a final concentration of 50 mmol / L, 6-methyl-5-hepten-2-ol with a final concentration of 15 mmol / L, and the rest was sodium hydrogen phosphate-sodium dihydrogen phosphate buffer (50 mmol / L, pH 8.0). The reaction system was reacted at 37 °C for 12 h. After the reaction, 20 μL of 250 mmol / L n-decanol was added as an internal standard, and then extracted with 2000 μL of n-butanol. 500 μL of the extract was taken for gas phase detection. The results are shown in Table 10.

[0057] The gas phase detection conditions for 6-methyl-5-hepten-2-ol were as follows: The gas chromatography column was HP-5 (30 m, 0.32 mm, 0.25 μm), held at 60 °C for 1 min, heated to 80 °C at 6 °C / min, then heated to 120 °C at 3 °C / min, and finally heated to 250 °C at 12 °C / min. The injection volume for each time was 1 μL, the carrier gas was nitrogen, and the flow rate was 1 mL·min -1 , and the split ratio was 50:1. The injection port temperature was 250 °C, and the FID temperature was 260 °C.

[0058] The total conversion rate C(%) = the number of moles of the total product × 100 / the number of moles of the initial substrate; the yield Y(%) = the number of moles of product A × 100 / the number of moles of the initial substrate; the selectivity S(%) = the number of moles of product A × 100 / the number of moles of the total product.

[0059] S5: Oxidation reaction of dihydrolinalool: The total volume of the reaction system was 1 mL, including: 114.3 mg of GOD / MsAcT cross-linked enzyme aggregates, glycerol triacetate with a final concentration of 50 mmol / L, glucose with a final concentration of 50 mmol / L, dihydrolinalool with a final concentration of 15 mmol / L, and the rest was sodium hydrogen phosphate-sodium dihydrogen phosphate buffer (50 mmol / L, pH 8.0). The reaction system was reacted at 37 °C for 24 h. After the reaction, 20 μL of 250 mmol / L n-decanol was added as an internal standard, and then extracted with 2000 μL of n-butanol. 500 μL of the extract was taken for gas phase detection. The results are shown in Table 10.

[0060] The gas phase detection conditions for dihydrolinalool were as follows: The gas chromatography column was HP-5 (30 m, 0.32 mm, 0.25 μm), held at 60 °C for 1 min, heated to 80 °C at 6 °C / min, then heated to 120 °C at 3 °C / min, and finally heated to 250 °C at 12 °C / min. The injection volume for each time was 1 μL, the carrier gas was nitrogen, and the flow rate was 1 mL / min, and the split ratio was 50:1. The injection port temperature was 250 °C, and the FID temperature was 260 °C.

[0061] The overall conversion rate C (%) = the number of moles of the total product × 100 / the number of moles of the initial substrate; the yield Y (%) = the number of moles of product A × 100 / the number of moles of the initial substrate; the selectivity S (%) = the number of moles of product A × 100 / the number of moles of the total product.

[0062] Table 10 Results of GOD / MsAcT cross-linked enzyme aggregates catalyzing different enol oxidation reactions

Claims

1. A method for preparing a glucose oxidase / acyltransferase cross-linked enzyme aggregate, characterized in that: The following steps are involved: 1) Preparation of Fe3O4@SiO2 magnetic nanoparticles; 2) Preparation of amino-functionalized Fe3O4@SiO2 magnetic nanoparticles; 3) Glucose oxidase and acyltransferase were immobilized on amino-functionalized Fe3O4@SiO2 magnetic nanoparticles by glutaraldehyde cross-linking to obtain glucose oxidase / acyltransferase cross-linked enzyme aggregates.

2. The preparation method according to claim 1, characterized in that: The step 1) is specifically as follows: ultrasonically dispersing 0.8 g of nano-Fe3O4 in a mixed solution consisting of 36 mL of anhydrous ethanol and 4 mL of distilled water, adding 1.6 mL of tetraethyl orthosilicate, and then adding PEG1000 with a final concentration of 1 mg / mL, and then adding 10 mL of 25 wt% ammonia water under stirring, stirring at room temperature for 12 hours, and then centrifuging to collect the precipitate, washing it with distilled water and anhydrous ethanol respectively, and then drying it at 60° C. overnight to obtain Fe3O4@SiO2 magnetic nanoparticles.

3. The preparation method according to claim 1, characterized in that: The step 2) is specifically as follows: ultrasonically disperse 0.25 g of Fe3O4@SiO2 magnetic nanoparticles in 20 mL of anhydrous ethanol, add PEG1000 with a final concentration of 1 mg / mL, adjust the pH to 4-8, add 0.5 mL of 3-aminopropyltriethoxysilane, react on a shaker at 20-40° C. for 12 h, then centrifuge, collect the precipitate, wash it with single distilled water and anhydrous ethanol respectively, and then dry it at 60° C. overnight to obtain amino-functionalized Fe3O4@SiO2 magnetic nanoparticles.

4. The preparation method according to claim 3, characterized in that: The step 2) is specifically as follows: ultrasonically disperse 0.25 g of Fe3O4@SiO2 magnetic nanoparticles in 20 mL of anhydrous ethanol, add PEG1000 with a final concentration of 1 mg / mL, adjust the pH to 4, add 0.5 mL of 3-aminopropyltriethoxysilane, react on a shaker at 40° C. for 12 h, then centrifuge, collect the precipitate, wash it with single distilled water and anhydrous ethanol respectively, and then dry it at 60° C. overnight to obtain amino-functionalized Fe3O4@SiO2 magnetic nanoparticles.

5. The preparation method according to claim 1, characterized in that: The step 3) is specifically as follows: 100 mg of amino-functionalized Fe3O4@SiO2 magnetic nanoparticles are ultrasonically dispersed in 1 mL of a pH 7.0, 50 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, glutaraldehyde with a final concentration of 5-80 mmol / L is added, the mixture is reacted at 25°C and 220 rpm for 1.5 h, magnetically separated, washed with a pH 7.0, 50 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, and then resuspended in 900 μL of a pH 7.0, 50 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to obtain a resuspended solution; 100 μL of a GOD / MsAcT mixed aqueous solution with a total protein content of 10 mg / mL is added to the obtained resuspended solution, the mixture is reacted at 25°C and 150 rpm for 1.5 h, magnetically separated, washed with a pH 7.0, 50 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to obtain a glucose oxidase / acyltransferase cross-linked enzyme aggregate.

6. The preparation method according to claim 5, characterized in that: The step 3) is specifically as follows: 100 mg of amino-functionalized Fe3O4@SiO2 magnetic nanoparticles are ultrasonically dispersed in 1 mL of a pH 7.0, 50 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, glutaraldehyde with a final concentration of 20 mmol / L is added, the mixture is reacted at 25°C and 220 rpm for 1.5 h, magnetically separated, washed with a pH 7.0, 50 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, and then resuspended in 900 μL of a pH 7.0, 50 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to obtain a resuspended solution; 100 μL of a GOD / MsAcT mixed aqueous solution with a total protein content of 10 mg / mL is added to the obtained resuspended solution, the mixture is reacted at 25°C and 150 rpm for 1.5 h, magnetically separated, washed with a pH 7.0, 50 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to obtain glucose oxidase / acyltransferase cross-linked enzyme aggregates.

7. The preparation method according to claim 5, characterized in that: The enzyme activity ratio of GOD:MsAcT in the GOD / MsAcT mixed aqueous solution is 50:

1.

8. A glucose oxidase / acyltransferase cross-linked enzyme aggregate, characterized in that: The glucose oxidase / acyltransferase cross-linked enzyme aggregate is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the glucose oxidase / acyltransferase cross-linked enzyme aggregate as claimed in claim 8 in catalyzing the oxidation reaction of enol.

10. The use according to claim 9, characterized in that: The enols include citronellol, 6-methyl-5-heptene-2-ol, and dihydrolinalool.

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