Immobilized glycosidase, preparation method thereof and application of immobilized glycosidase in preparation of resveratrol

Abstract

The invention discloses a preparation method of immobilized glycosidase and application of the immobilized glycosidase in preparation of resveratrol, and belongs to the technical field of preparation of immobilized enzymes. The specific preparation method comprises the following steps: (1) carrying out amination modification on glycosidase by using ethylenediamine and N-(3-dimethylaminopropyl)-N '-ethylcarbodiimide hydrochloride, so as to obtain aminated glycosidase; (2) dissolving an HOF material containing pyrenyl tetracarboxylic acid and a derivative structure thereof in N, N-dimethylformamide to activate carboxyl of the HOF material; and (3) rapidly mixing the aminated glycosidase and the carboxyl-activated HOF material in a stirring state, stirring, standing, centrifugally separating and precipitating, and re-dissolving the precipitate in deionized water to obtain the immobilized enzyme. The preparation method of the immobilized glycosidase has the advantages of mild conditions, rapid immobilization and the like, and the immobilized glycosidase still has catalytic activity of 60% or above after being repeatedly used for 10 times and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of immobilized glycosidase, and mainly relates to an immobilized glycosidase and a preparation method thereof and application in the preparation of resveratrol. Background Art

[0002] Resveratrol is a non-flavonoid polyphenol compound with significant antioxidant activity. It is widely found in plants such as Japanese knotweed, grapes, and peanuts. It plays an important role in preventing cancer, protecting the cardiovascular and nervous systems, enhancing immunity, and prolonging life. However, resveratrol exists primarily in natural plants as its glycoside, polydatin, which has lower bioavailability than resveratrol. Therefore, degrading the glycoside from polydatin to produce resveratrol is an effective method for obtaining resveratrol. Currently, the synthesis of resveratrol is mainly carried out through chemical methods, but this method has disadvantages such as harsh reaction conditions, high environmental pollution, cumbersome procedures, and low yield. In contrast, enzymatic methods have attracted widespread attention in recent years due to their environmental friendliness, safety, and efficiency.

[0003] Glycosidases, also known as glycoside hydrolases (EC 3.2.1), are enzymes that hydrolyze glycosidic bonds. Glycosidic bonds are chemical bonds that connect sugar molecules (monosaccharides or polysaccharides) to other molecules (such as sugars, proteins, and lipids). Glycosidases play a crucial role in glycosidic bond modification reactions, encompassing a wide range of applications in food, chemical engineering, biopharmaceuticals, and other fields, and hold a significant position in the global enzyme market.

[0004] However, free glycosidases have problems such as difficulty in recycling, poor stability and high cost, which limit their industrial application. Free glycosidases are easily inactivated in low water activity environments, have poor stability in organic solvent systems, and are easily induced by solvents to change their structure or unfold, resulting in inactivation or denaturation. In addition, the separation and purification of free glycosidases is costly and time-consuming, and it is difficult to recycle them after the reaction is completed, which increases the cost of use. Moreover, the catalytic efficiency in some reactions is also less than ideal.

[0005] Enzyme immobilization technology, a method for improving enzyme stability, reusability, and ease of use by adsorbing or encapsulating enzymes into supports, has garnered widespread attention in recent years. Currently, a wide variety of enzyme immobilization supports exist, including macroporous resins, chitosan supports, metal-organic frameworks (MOFs), and covalent organic frameworks (COFs). However, these supports present challenges in enzyme immobilization, such as long immobilization times, demanding synthesis conditions, and the presence of toxic metal ions. Hydrogen-bonded organic frameworks (HOFs) are a novel porous material. These materials are typically highly ordered porous structures formed by the self-assembly of organic molecules through hydrogen bonds. They offer unique advantages such as mild crystallization conditions, strong designability, high porosity, and excellent chemical stability. Their highly ordered pore structure provides an optimal spatial environment for enzyme immobilization, effectively protecting the enzyme's active sites and minimizing enzyme activity loss during the immobilization process. Furthermore, the chemical stability of HOFs enables them to remain stable under a variety of reaction conditions, thereby extending the lifespan of immobilized enzymes.

[0006] However, there is currently no research on the preparation of immobilized glycosidases using hydrogen-bonded organic framework (HOF) materials. How to fully utilize the characteristics of HOF materials to develop an efficient and stable immobilized glycosidase system is of great significance for expanding the application range of glycosidases and improving their industrial application value. Based on this technical background, the present invention aims to provide an innovative method for preparing immobilized glycosidases using hydrogen-bonded organic framework (HOF) materials and an immobilized glycosidase product, which realizes efficient immobilization of glycosidases through modification and hydrogen bond self-assembly of glycosidases while maintaining high catalytic activity and stability, providing a new technical approach for the application of glycosidases in industrial production. Summary of the Invention

[0007] The present invention provides a method for preparing a novel immobilized glycosidase using a hydrogen-bonded organic framework (HOF) material, and its application in the preparation of resveratrol. The glycosidase is chemically modified by amination, and then self-assembled on the surface of the aminated glycosidase using a HOF material containing a pyrenyltetracarboxylic acid and its derivative structure (such as 1,3,6,8-tetra(p-benzoyl)pyrene (H4TBAPy)) to obtain an immobilized glycosidase. The immobilization process is rapid and efficient, under mild conditions, and the immobilized glycosidase has good reusability and stability, enabling efficient glycosylation of resveratrol.

[0008] The present invention adopts the following technical solutions:

[0009] The present invention uses an aminated glycosidase as the core, introduces a carboxyl-activated HOF material onto its surface, and rapidly assembles the enzyme onto the surface to produce an immobilized glycosidase. The aminated glycosidase has numerous amino groups on its surface, which facilitates hydrogen bonding with the carboxyl groups on the activated HOF material, allowing for rapid self-assembly within a short period of time. Furthermore, the immobilized glycosidase prepared in this invention exhibits excellent reusability and stability, and demonstrates efficient glycosyl conversion to resveratrol.

[0010] A method for preparing immobilized glycosidase comprises the following steps:

[0011] (1) Under acidic or neutral conditions, the glycosidase is dissolved in ethylenediamine, and then N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is added to modify the glycosidase by amination, and dialyzed to obtain an aminated glycosidase with a rich surface amino group;

[0012] (2) Under ultrasonic conditions, the HOF material containing the structure of pyrenyltetracarboxylic acid and its derivatives is dissolved in N,N-dimethylformamide to obtain a carboxyl-activated HOF material;

[0013] (3) Under stirring conditions, the aminated glycosidase and the carboxyl-activated HOF material are quickly stirred and mixed, allowed to stand and then centrifuged, and the precipitate is redissolved in deionized water to obtain the immobilized glycosidase.

[0014] Furthermore, in step (1), the acidic or neutral pH range is 4.0-7.0, preferably pH 4.5.

[0015] Furthermore, in step (1), the glycosidase is derived from one of filamentous fungi, yeast, and actinomycetes, and has a concentration of 0.5-5 mg / mL.

[0016] Furthermore, in step (1), the concentration of ethylenediamine is 0.1M-1M, and the concentration of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 10mM-50mM.

[0017] Furthermore, in step (1), the amination modification is carried out under ice bath conditions with stirring for 0.5-3 h.

[0018] Furthermore, in step (1), the molecular cutoff rate of the dialysis bag used for dialysis is 8000-14000 kDa.

[0019] Furthermore, in step (2), the HOF material containing the structure of pyrenyltetracarboxylic acid and its derivatives includes one of 1,3,6,8-tetracarboxypyrene, 1,3,6,8-tetra(p-benzoic acid)pyrene, and 1,3,6,8-tetra(6-carboxynaphthalen-2-yl)pyrene.

[0020]

[0021] More preferably, the HOF material of the pyrene tetracarboxylic acid and its derivative structure of the present invention is 1,3,6,8-tetrakis(p-benzoic acid)pyrene.

[0022] Furthermore, the mass volume ratio of the HOF material containing the structure of pyrenyltetracarboxylic acid and its derivatives to N,N-dimethylformamide is 1-20 mg / mL.

[0023] Furthermore, in step (3), the aminated glycosidase and the carboxyl-activated HOF material are stirred and mixed at room temperature for 5-10 minutes and allowed to stand for 0.5-2 hours.

[0024] More preferably, the stirring time is 5 minutes and the standing time is 0.5 hours.

[0025] Furthermore, in step (3), the centrifugation condition is 12840g-13300g, the centrifugation time is 6-10min, and after the centrifugation is completed, deionized water is added to wash the precipitate, and the centrifugation is repeated 3 times, and the final dispersion is in deionized water.

[0026] The activity determination method of the immobilized glycosidase obtained by the above method is as follows: 1 mL of 0.1 M citric acid buffer (pH 4.0) and 400 μL of pNPG were added to a 2 mL centrifuge tube, and the mixture was incubated at 50°C for 10 minutes, followed by the addition of 400 μL of immobilized glycosidase. After reacting in a water bath for 5 mL, the immobilized enzyme was separated by centrifugation. 500 μL of the supernatant was added to 500 μL of 1 M sodium carbonate, and the absorbance was measured at 405 nm.

[0027] The activity recovery rate of the immobilized glycosidase prepared by the method of the present invention is 93.3%, and after repeated catalysis for 10 times, the activity is still 59.6%, and the immobilized glycosidase has better stability and reusability than the free enzyme.

[0028] The present invention also provides the use of the above-mentioned immobilized glycosidase in the preparation of resveratrol, and the method is as follows:

[0029] The substrate polydatin, a buffer, and the prepared immobilized glycosidase are mixed to perform a glycosyl hydrolysis reaction. The buffer is a citric acid buffer with a pH of 3-6 or a phosphate buffer with a pH of 6-8. The reaction temperature is 30-60° C., the reaction pH is 3-8, and the reaction time is 0.5-5 hours.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0031] (1) The present invention provides a novel method for preparing immobilized glycosidase, wherein ethylenediamine and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride are used as amination reagents to modify the glycosidase by amination, and the glycosidase is chemically modified by amination. After modification, HOF materials containing pyrene tetracarboxylic acid and its derivative structures (such as 1,3,6,8-tetra(p-benzoyl)pyrene) are self-assembled on the surface of the aminated glycosidase to obtain immobilized glycosidase.

[0032] (2) The preparation method of the present invention provides mild immobilization conditions, allowing immobilization to be performed at room temperature. Furthermore, the immobilization time is short, requiring only 30 minutes. The obtained immobilized glycosidase exhibits improved reactivity at different temperatures and pH levels and better storage stability.

[0033] (3) Free glycosidases are often expensive, non-reusable, and difficult to separate. The immobilized glycosidase prepared in the present invention has good reusability and still has more than 60% catalytic activity after 10 catalytic reactions. It can be separated from the system by centrifugation.

[0034] (4) The immobilized glycosidase obtained from the HOF material 1,3,6,8-tetra(p-benzoyl)pyrene of the present invention achieved an immobilization efficiency of up to 85.3% and an enzyme activity recovery rate of 93.3%. The preparation method of the present invention is also applicable to other HOF materials with similar structures, including but not limited to 1,3,6,8-tetracarboxypyrene and 1,3,6,8-tetra(6-carboxynaphthalen-2-yl)pyrene. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a structural diagram of the basic building block of the HOF material 1,3,6,8-tetrakis(p-benzoic acid)pyrene used in the present invention.

[0036] Figure 2 Graph showing the immobilization efficiency and activity recovery rate of samples obtained in Examples 1-3 and Comparative Example 1 of the present invention.

[0037] Figure 3 The relative activities of the immobilized glycosidase and free glycosidase prepared in Example 1 of the present invention at different temperatures.

[0038] Figure 4 The relative activities of the immobilized glycosidase and free glycosidase prepared in Example 1 of the present invention at different pH values.

[0039] Figure 5 This is the change in storage activity of the immobilized glycosidase and free glycosidase prepared in Example 1 of the present invention at 4°C.

[0040] Figure 6The relative activity of the immobilized glycosidase prepared in Example 1 of the present invention under different reuse times.

[0041] Figure 7 This is a liquid chromatogram of a mixture of commercially available polydatin and resveratrol standards.

[0042] Figure 8 The figure is a liquid chromatogram of the immobilized glycosidase of the present invention before reaction in the application of preparing resveratrol.

[0043] Figure 9 The figure is a liquid chromatogram after the immobilized glycosidase of the present invention reacts in the application of preparing resveratrol. DETAILED DESCRIPTION

[0044] The specific implementation of the present invention will be further described below in conjunction with the examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0045] In the embodiment of the present invention, the glycosidase is a commercial glycosidase derived from filamentous fungi, named Aromase H2, and purchased from Amano Enzyme Co., Ltd.

[0046] The immobilization efficiency and activity recovery rate of the immobilized glycosidase in the present invention are calculated according to formulas 1 and 2, respectively:

[0047] Formula 1:

[0048] Formula 2:

[0049] In formula 1, C1 and V1 are the protein concentration and volume before immobilization, respectively; C2 and V2 are the protein concentration and volume of the supernatant after immobilization, respectively;

[0050] A in Formula 2 immobilized is the immobilized enzyme activity, A initial is the free enzyme activity before immobilization.

[0051] Figure 1 This is the basic unit structure of 1,3,6,8-tetrakis(p-benzoyl)pyrene, a HOF material used in an embodiment of the present invention (gray circles represent carbon atoms, white circles represent hydrogen atoms, and red circles represent hydrogen atoms). Its terminal ends contain four symmetrical carboxyl groups that can form hydrogen bonds with amino groups on the surface of glycosidase, thereby achieving glycosidase immobilization.

[0052] Example 1

[0053] Preparation of immobilized glycosidase

[0054] Step (1): prepare a 1M ethylenediamine solution, adjust its pH to 4.5 using 1M hydrochloric acid, add glycosidase to the system to a final concentration of 2 mg / mL, add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride to a final concentration of 10 mM after ultrasonic dissolution, stir for 2 hours under ice bath conditions, boil a dialysis bag with a molecular weight cutoff of 8000-14000 kDa in boiling water for 10 minutes, transfer the liquid in the system to the dialysis bag after stirring, dialyze at 4°C, replace distilled water every 6 hours, and leave overnight after the last water change. After the dialysis is completed, transfer the enzyme solution in the dialysis bag to a centrifuge tube to obtain the aminated glycosidase.

[0055] Step (2): Weigh 12 mg of 1,3,6,8-tetrakis(p-benzoic acid)pyrene (H4TBAPy), add 1.2 mL of anhydrous N,N-dimethylformamide, and dissolve it with ultrasound to obtain a carboxyl-activated HOF material.

[0056] Step (3): While stirring, the aminated glycosidase from step (1) was quickly poured into the carboxyl-activated HOF material from step (2), stirred for 5 minutes, and then allowed to stand at room temperature for 30 minutes. After standing, the mixture was centrifuged at 13300g for 6 minutes. After each centrifugation, the supernatant and precipitate were separated. 5 mL of deionized water was added to wash the precipitate, and the centrifugation was continued under the above centrifugation conditions. The supernatant of each centrifugation was collected. After repeating this process three times, the precipitate was dispersed in deionized water for the last time, sonicated, and dissolved to obtain the immobilized glycosidase prepared. The immobilized glycosidase was stored at 4°C for later use.

[0057] Example 2

[0058] Preparation of immobilized glycosidase

[0059] Step (1): prepare a 1M ethylenediamine solution, adjust its pH to 7.0 using 1M hydrochloric acid, add glycosidase to the system to a final concentration of 2 mg / mL, add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride to a final concentration of 10 mM after ultrasonic dissolution, stir for 2 hours under ice bath conditions, boil a dialysis bag with a molecular weight cutoff of 8000-14000 kDa in boiling water for 10 minutes, transfer the liquid in the system to the dialysis bag after stirring, dialyze at 4°C, replace distilled water every 6 hours, and leave overnight after the last water change. After the dialysis is completed, transfer the enzyme solution in the dialysis bag to a centrifuge tube to obtain aminated glycosidase.

[0060] Step (2): Weigh 12 mg of 1,3,6,8-tetrakis(p-benzoic acid)pyrene (H4TBAPy), add 1.2 mL of anhydrous N,N-dimethylformamide, and dissolve under ultrasound to obtain a carboxyl-activated HOF material.

[0061] Step (3): While stirring, the aminated glycosidase from step (1) was quickly poured into the carboxyl-activated HOF material from step (2), stirred for 5 minutes, and then allowed to stand at room temperature for 30 minutes. After standing, the mixture was centrifuged at 13300g for 6 minutes. After each centrifugation, the supernatant and precipitate were separated. 5 mL of deionized water was added to wash the precipitate, and the centrifugation was continued under the above centrifugation conditions. The supernatant of each centrifugation was collected. After repeating this process three times, the precipitate was dispersed in deionized water for the last time, sonicated, and dissolved to obtain the immobilized glycosidase prepared. The immobilized glycosidase was stored at 4°C for later use.

[0062] Example 3

[0063] Preparation of immobilized glycosidase

[0064] Step (1): prepare a 1M ethylenediamine solution, adjust its pH to 4.5 using 1M hydrochloric acid, add glycosidase to the system to a final concentration of 0.5 mg / mL, dissolve it with ultrasound, add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride to a final concentration of 10 mM, stir for 2 hours under ice bath conditions, boil a dialysis bag with a molecular weight cutoff of 8000-14000 kDa in boiling water for 10 minutes, transfer the liquid in the system to the dialysis bag after stirring, dialyze at 4°C, replace distilled water every 6 hours, and leave it overnight after the last water change. After the dialysis is completed, transfer the enzyme solution in the dialysis bag to a centrifuge tube to obtain the aminated glycosidase.

[0065] Step (2): Weigh 12 mg of 1,3,6,8-tetrakis(p-benzoic acid)pyrene (H4TBAPy), add 1.2 ml of anhydrous N,N-dimethylformamide, and dissolve it with ultrasound to obtain a carboxyl-activated HOF material.

[0066] Step (3): While stirring, the aminated glycosidase from step (1) was quickly poured into the carboxyl-activated HOF material from step (2), stirred for 5 minutes, and then allowed to stand at room temperature for 30 minutes. After standing, the mixture was centrifuged at 13300g for 6 minutes. After each centrifugation, the supernatant and precipitate were separated. 5 mL of deionized water was added to wash the precipitate, and the centrifugation was continued under the above centrifugation conditions. The supernatant of each centrifugation was collected. After repeating this process three times, the precipitate was dispersed in deionized water for the last time, sonicated, and dissolved to obtain the immobilized glycosidase prepared. The immobilized glycosidase was stored at 4°C for later use.

[0067] Example 4

[0068] Before immobilization, 50 μL of the aminated glycosidase solution in Examples 1, 2, and 3 was taken, and the supernatant collected from each centrifugation in Examples 1, 2, and 3 was taken. The protein concentration in the supernatant before immobilization and after centrifugation was determined using the Bradford method. The immobilization efficiency was calculated using Formula 1. The measured immobilization efficiencies of Examples 1, 2, and 3 were 85.3%, 17.9%, and 49.9%, respectively.

[0069] Example 5

[0070] A free glycosidase solution of the same concentration as in Example 1 was prepared. 1 mL of 0.1 M citric acid buffer at pH 4.0 and 400 μL of 1 mM p-nitrophenol-β-D-glucoside (p-NPG) were incubated at the optimal temperature for 10 minutes, followed by the addition of 400 μL of the free glycosidase solution or the immobilized enzyme obtained in Example 1. The reaction was continued at the optimal temperature for five minutes. The free glycosidase was quickly inactivated by placing it in boiling water. The immobilized glycosidase was centrifuged to separate the precipitate and supernatant, and 500 μL of 1 M Na2CO3 was added to each of the precipitates and supernatants. The absorbance was then measured at 405 nm using a spectrophotometer. The enzyme activity (U) was defined as the amount of enzyme required to produce 1 μmol pNP per minute under the above reaction conditions. The activity recovery rate was calculated using Formula 2. The activity recovery rates of Examples 1, 2, and 3 were 93.3%, 44.3%, and 46.8%, respectively.

[0071] Comparative Example 1

[0072] Prepare an unaminated free glycosidase solution of the same concentration as in Example 1, weigh 12 mg of 1,3,6,8-tetrakis(p-benzoyl)pyrene (H4TBAPy), add 1.2 mL of anhydrous N,N-dimethylformamide, and dissolve it with ultrasound. Pour the prepared free glycosidase solution into it quickly under stirring, stir for 5 minutes, and then let it stand at room temperature for 30 minutes. After standing, centrifuge at 13300g for 6 minutes. After each centrifugation, separate the supernatant and precipitate. Add 5 mL of deionized water to wash the precipitate and continue centrifugation under the above centrifugation conditions, collecting the supernatant of each centrifugation. Repeat 3 times, disperse the precipitate in deionized water for the last time, dissolve it with ultrasound, and store it at 4°C for use. The immobilization efficiency and activity recovery rate were determined according to Examples 3 and 4. The measured immobilization efficiency was 13.5% and the activity recovery rate was 3.1%.

[0073] Figure 2Figure 1 is the immobilization efficiency and activity recovery rate of the samples obtained in Examples 1-3 and Comparative Example 1. It can be seen that the immobilized glycosidase in Example 1 has an immobilization efficiency of 85.3% and an activity recovery rate of 93.3%. When the pH is 4.5, the degree of amination modification of the free enzyme is the best, the immobilization efficiency is the highest, and the material is most bound to the enzyme surface, so the immobilization efficiency and activity recovery rate are the best. When the pH is changed to 7, the amination modification effect of the immobilized glycosidase in Example 2 is poor, resulting in less material assembly on the enzyme surface. The enzyme concentration in Example 3 is low, and the content of the immobilized enzyme is low. These reasons lead to a decrease in the immobilization efficiency and activity recovery rate of Examples 2 and 3. In Comparative Example 1, when free enzyme is used for immobilization, the lowest activity recovery rate and immobilization efficiency are achieved because the free enzyme has few amino groups on the surface, resulting in low enzyme immobilization efficiency.

[0074] Example 6

[0075] A system containing 1 mL of 0.1 M citric acid buffer at pH 4.0 and 400 μL of 1 mM p-NPG was incubated at 30-90°C (30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C, respectively) for 10 minutes, and then 400 μL of free glycosidase solution or the immobilized glycosidase obtained in Example 1 was added. After 5 minutes of reaction, the free glycosidase was quickly placed in boiling water for inactivation. The immobilized glycosidase was centrifuged to separate the precipitate and supernatant, and 500 μL of 1 M Na2CO3 was added to each of them for color development. The absorbance was measured at 405 nm using a spectrophotometer. The results are shown in FIG. Figure 3 As shown, the activity of the immobilized enzyme increases with increasing temperature, reaching its peak at 50°C. As the temperature continues to rise, the enzyme activity is inhibited. Most importantly, the immobilized glycosidase prepared by the method of the present invention enhances the activity of the free enzyme at different temperatures.

[0076] Example 7

[0077] A system containing 1 ml of 0.1 M buffer (pH 3-6: citrate buffer, pH 6-8: phosphate buffer, pH 8-9: Tris-HCl buffer) with different pH values ​​of 3-9 (3, 4, 5, 6, 7, 8, 9, respectively) and 400 μL of 1 mM p-NPG was incubated at the optimal temperature for free enzyme and immobilized enzyme for 10 min, and then 400 μL of free glycosidase solution or the immobilized enzyme obtained in Example 1 was added. After reacting for 5 minutes, the free glycosidase was quickly inactivated by placing it in boiling water. The immobilized glycosidase was centrifuged to separate the precipitate and supernatant, and 500 μL of 1 M Na2CO3 was added to each of them for color development. The absorbance was measured at 405 nm using a spectrophotometer. The results are shown in FIG. Figure 4As shown, the free enzyme is an acidic enzyme, and its activity decreases as the pH increases, significantly affecting its activity. This trend remains unchanged after immobilization. Like the free enzyme, the immobilized glycosidase has optimal activity at pH 4, but regardless of pH, the immobilized glycosidase has higher activity than the free enzyme.

[0078] Example 8

[0079] The immobilized glycosidase prepared in Example 1 and the freshly prepared free glycosidase were stored at 4°C and the enzyme activity was measured every 7 days according to the method described in Example 4. The activity measured on day 0 was taken as 100% to measure the storage stability. Figure 5 After 8 weeks of storage, the immobilized glycosidase maintained 94.04% of its activity, showing good storage stability.

[0080] Example 9

[0081] 1 mL of 0.1 M citric acid buffer at pH 4.0 and 400 μL of 1 mM p-NPG were incubated at 50°C for 10 minutes, followed by the addition of 400 μL of the immobilized glycosidase obtained in Example 1. After five minutes of reaction, the precipitate and supernatant were separated by centrifugation. 500 μL of the supernatant was added to 500 μL of 1 M Na2CO3 for color development, and the absorbance was measured at 405 nm using a spectrophotometer. The precipitate was washed twice with deionized water and redissolved in 400 μL of deionized water. The above process was repeated 10 times, with the activity measured for the first time being 100%. The results are as follows: Figure 6 As shown in the figure, more than 60% of the catalytic activity was retained after 10 catalytic cycles.

[0082] Example 10

[0083] The immobilized glycosidase prepared by the method of Example 1 was used to prepare resveratrol:

[0084] 12 mg of polydatin was used as the substrate and dissolved in 400 μL of DMSO. 1.4 mL of pH 3.0 citrate buffer was added, and finally 200 μL of the prepared immobilized glycosidase was added. The reaction was shaken in a constant temperature shaking incubator (50°C, 120 rpm) for 2 hours. The detection method was high-performance liquid chromatography (HPLC) using an Agilent Zorbax SB-C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase consisted of 25% (v / v) acetonitrile-water solution (containing 0.1% (v / v) formic acid). The elution mode was isocratic, the flow rate was 0.9 mL / min, the detection wavelength was 305 nm, the injection volume was 20 μL, and the column temperature was 30°C.

[0085] Figure 7This is the liquid chromatogram of a mixture of commercially available polydatin and resveratrol standards. It can be seen that polydatin peaks at approximately 4.2 minutes, and resveratrol peaks at approximately 12 minutes. Figure 8 This is the liquid chromatogram of the immobilized glycosidase in Example 10 before the reaction in the preparation of resveratrol. Only the peak of the substrate polydatin can be detected. Figure 9 This is a liquid chromatogram of the immobilized glycosidase after the reaction in the preparation of resveratrol in Example 10. The disappearance of the polydatin substrate peak and the appearance of the product resveratrol peak indicate that the immobilized glycosidase prepared by the method of the present invention successfully hydrolyzes polydatin to produce resveratrol.

[0086] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing immobilized glycosidase, characterized in that: The following steps are involved: (1) Under acidic or neutral conditions, the glycosidase is dissolved in ethylenediamine, and then N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is added to modify the glycosidase by amination, and dialyzed to obtain an aminated glycosidase with a rich surface amino group; (2) Under ultrasonic conditions, the HOF material containing the structure of pyrenyltetracarboxylic acid and its derivatives is dissolved in N,N-dimethylformamide to obtain a carboxyl-activated HOF material; (3) Under stirring conditions, the aminated glycosidase and the carboxyl-activated HOF material are quickly stirred and mixed, allowed to stand and then centrifuged, and the precipitate is redissolved in deionized water to obtain the immobilized glycosidase.

2. The method for preparing the immobilized glycosidase according to claim 1, wherein: In step (1), the acidic or neutral pH range is 4.0-7.

0.

3. The method for preparing the immobilized glycosidase according to claim 1, characterized in that: In step (1), the glycosidase is derived from one of filamentous fungi, yeast, and actinomycetes, and the concentration of the glycosidase is 0.5-5 mg / mL.

4. The method for preparing the immobilized glycosidase according to claim 1, wherein: In step (1), the concentration of ethylenediamine is 0.1M-1M, and the concentration of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 10mM-50mM.

5. The method for preparing the immobilized glycosidase according to claim 1, wherein: In step (1), the amination modification is carried out under ice bath conditions with stirring for 0.5-3 h.

6. The method for preparing the immobilized glycosidase according to claim 1, characterized in that: In step (2), the HOF material containing the structure of pyrenyltetracarboxylic acid and its derivatives includes one of 1,3,6,8-tetra(p-benzoic acid)pyrene, 1,3,6,8-tetracarboxypyrene, and 1,3,6,8-tetra(6-carboxynaphthalen-2-yl)pyrene.

7. The method for preparing the immobilized glycosidase according to claim 1, characterized in that: In step (2), the mass volume ratio of the HOF material containing the structure of pyrenyltetracarboxylic acid and its derivatives to N,N-dimethylformamide is 1-20 mg / mL.

8. The method for preparing the immobilized glycosidase according to claim 1, characterized in that: In step (3), the aminated glycosidase and the carboxyl-activated HOF material are stirred and mixed at room temperature for 5-10 minutes and for 0.5-2 hours.

9. The immobilized glycosidase obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the immobilized glycosidase according to claim 9 in the preparation of resveratrol, characterized in that: The substrate polydatin, buffer solution and immobilized glycosidase are mixed and reacted at a temperature of 30-60° C. and a pH of 3-8 for 0.5-5 hours.

Images