Optically controlled dual-enzyme activity zein-based mimic enzyme and preparation method thereof

Abstract

The application is suitable for the field of analog enzyme technology, and provides a light-controlled zein-based dual-enzyme-activity analog enzyme and a preparation method thereof.The preparation method comprises the following steps: under water bath conditions, dissolving zein in an alkaline aqueous solution and incubating; adding a hydroxylamine solution to the solution obtained in step S1 and continuing to react; and after the reaction is completed, obtaining the light-controlled zein-based dual-enzyme-activity analog enzyme.The application uses zein as a raw material, is modified by hydroxylamine, does not need any metal component to participate, has light-controlled switchable dual-enzyme activity, exhibits oxidase-like activity under visible light irradiation, presents peroxidase-like activity in dark conditions, and can realize reversible switching of the two enzyme activities by regulating light, effectively overcomes the problems that traditional artificial analog enzymes depend on metal ions, multiple enzyme activities coexist, interfere with each other and are difficult to accurately control, can be used for colorimetric detection of total antioxidant capacity, can be used for colorimetric detection of glucose in cascade reaction with glucose oxidase, has the advantages of simple operation, rapid response, high sensitivity, good selectivity, low cost and the like, and has a good application prospect in the fields of food analysis, clinical diagnosis and the like.

Description

Technical Field

[0001] This invention belongs to the field of enzyme mimicry technology, and particularly relates to a zein-based enzyme mimicry with light-controlled dual enzyme activity and its preparation method. Background Technology

[0002] Natural enzymes possess highly efficient catalytic activity and play a crucial role in industrial production and analytical detection. However, the activity of natural enzymes is easily affected by environmental factors such as temperature and pH. Furthermore, the complex extraction and purification processes, demanding storage conditions, and difficulties in recovery severely limit their large-scale application in abiotic environments. Therefore, developing artificial enzyme mimics that can simulate the catalytic function of natural enzymes is currently a research hotspot. Compared to natural enzymes, mimics offer advantages such as superior catalytic stability, low preparation cost, and ease of large-scale production. Moreover, their catalytic activity can be rationally controlled through material design.

[0003] However, existing enzyme mimicry systems still suffer from the following problems: most mimicry rely on noble or transition metals, posing risks of metal leakage and potential biosafety issues; their synthesis requires harsh conditions, often necessitating high temperatures, organic solvents, or inert atmospheres; some mimicry possess multiple enzyme activities simultaneously, but these different activities may compete for the same substrate, leading to interference in catalytic pathways and making precise regulation difficult. In particular, the reversible switching regulation of oxidase-like and peroxidase-like activities remains a critical problem that urgently needs to be solved in the field of enzyme mimicry research. Therefore, developing a novel metal-free, easily synthesized, and light-controlled enzyme mimicry with switchable dual enzyme activities has significant theoretical and practical value. Summary of the Invention

[0004] The purpose of this invention is to provide a light-controlled dual-enzyme activity zein-based mimic enzyme and its preparation method, aiming to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] On one hand, the present invention provides a method for preparing a zein-based mimic enzyme with light-controlled dual-enzyme activity, comprising the following steps:

[0007] Step S1: Dissolve zein in an alkaline aqueous solution and incubate under water bath conditions;

[0008] Step S2: Add hydroxylamine solution to the solution obtained in step S1 and continue the reaction;

[0009] Step S3: After the reaction is complete, a zein-based mimic enzyme with light-controlled dual-enzyme activity is obtained.

[0010] On the other hand, the present invention provides a light-controlled dual-enzyme activity zein-based mimic enzyme, which is prepared by the above-described preparation method.

[0011] On the other hand, the present invention provides the application of a light-controlled dual-enzyme activity zein-based mimic enzyme in colorimetric detection.

[0012] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0013] This invention prepares a light-controlled dual-enzyme mimicking enzyme by reacting zein with hydroxylamine (the glutamine side chain amide group of zein reacts with hydroxylamine to generate a hydroxamic acid group). This mimicking enzyme exhibits oxidase-like activity under visible light irradiation and peroxidase-like activity in darkness. The two enzyme activities can be reversibly switched by light control. This mimicking enzyme does not contain any metal elements; its enzyme-like activity originates from the catalytic generation of superoxide anion radicals (·O2) from dissolved oxygen under visible light excitation. - );

[0014] This invention is the first to prepare a photosensitive enzyme with dual-enzyme activity using natural zein as raw material and hydroxylamine modification via a one-step aqueous-phase synthesis method. The reaction conditions are mild, requiring no organic solvents, precious metals, or inert atmosphere protection, resulting in low cost, simple operation, and easy large-scale production. The obtained photosensitive enzyme contains no metal elements, exhibits good biocompatibility, and has high safety.

[0015] The simulated enzyme provided by this invention exhibits oxidase-like activity under visible light irradiation and peroxidase-like activity under dark conditions. The reversible switching of the two enzyme activities can be achieved by a simple light switch, effectively solving the problem of mutual interference when multiple enzyme activities coexist.

[0016] The colorimetric detection method for total antioxidant capacity based on the photocontrolled oxidase activity of the enzyme mimicking this invention has a detection limit of 0.18 μM for ascorbic acid and a linear range of 0–30 μM. The glucose cascade colorimetric detection method based on its peroxidase-like activity has a detection limit of 2.7 μM for glucose and a linear range of 5–70 μM. The spiked recoveries of the two methods in actual fruit juice samples were 99.1%–102.5% and 99.1%–101.4%, respectively. Attached Figure Description

[0017] Figure 1 Fourier transform infrared spectra of ZE and zein provided in this embodiment of the invention;

[0018] Figure 2 The following are the UV-Vis spectra of ZE provided in the embodiments of the present invention: A is the UV-Vis spectra of ZE, TMB, zein and TMB under visible light irradiation; B is the UV-Vis spectra of ZE under visible light and dark environments.

[0019] Figure 3 The UV-Vis spectra of different catalytic systems for evaluating peroxidase activity provided in embodiments of the present invention;

[0020] Figure 4 The absorbance gradient of ZE at 652 nm was detected under conditions of sequentially turning visible light on and off, as provided in an embodiment of the present invention.

[0021] Figure 5 The results show the relative catalytic activity of ZE photo-controlled oxidase under different storage times provided in the embodiments of the present invention;

[0022] Figure 6 For the total antioxidant capacity detection results provided in the embodiments of the present invention, A is a curve of A0-A versus ascorbic acid (AA) concentration, and B is a linear range graph of total antioxidant capacity detection;

[0023] Figure 7 The glucose detection results provided in this embodiment of the invention are shown in Figure A, which is a curve of absorbance versus glucose concentration for glucose detection systems of different concentrations, and Figure B, which is a linear range graph of glucose detection. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0025] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0026] Example 1: A light-controlled dual-enzyme activity zein-based mimic enzyme, the preparation method of which includes the following steps:

[0027] Dissolve 2 mg of zein powder in an alkaline solution (NaOH, 0.04 M, 2.5 mL) and incubate with stirring in a 60°C water bath for 10 minutes. Then, add 2 mL of hydroxylamine solution (50 mM) and 0.5 mL of deionized water to the above solution to obtain a zein-based mimic enzyme (ZE) with light-controlled dual enzyme activity.

[0028] ZE was characterized using Fourier transform infrared spectroscopy, and the results are as follows: Figure 1 As shown, the results are displayed at approximately 1660 cm. -1 The appearance of a new absorption peak at this point is attributed to the C=O stretching vibration of hydroxamic acid, indicating the successful introduction of the hydroxamic acid group.

[0029] Example 2: Validation of the light-controlled dual-enzyme activity of a zein-based mimetic enzyme (ZE) with light-controlled dual-enzyme activity;

[0030] Mix 100 μL of ZE solution, 100 μL of TMB solution (0.1 mM), and 800 μL of acetate buffer (pH 4.0, 20 mM); divide the mixture into two groups: the first group was irradiated under a xenon lamp for 7 minutes, and the solution turned blue with a characteristic absorption peak at 652 nm; Figure 2 As shown in the control experiment, no color reaction was observed in the solution without ZE or light. In the second group, after adding 100 μL of H₂O₂ (100 mM) under dark conditions and reacting for 10 minutes, the solution turned blue and showed a characteristic absorption peak at 652 nm. Similarly, Figure 3 The control experiment showed that no color reaction occurred in the solution when there was no H2O2 present.

[0031] In addition, the absorbance at 652 nm was monitored by alternately turning the xenon lamp on and off, and the results are as follows. Figure 4 As shown, the absorbance increases rapidly during the light phase and remains basically unchanged during the dark phase, indicating that the reversible switching of the two enzyme activities can be achieved by controlling the light.

[0032] ZE was stored at room temperature in the dark. After 42 days, its light-controlled oxidase activity was measured using the method described above. Figure 5 As shown, it still retains more than 60% of its initial catalytic activity.

[0033] Example 3: Application of light-controlled dual-enzyme activity zein-based mimic enzyme (ZE) in colorimetric detection:

[0034] The activity of light-controlled oxidases was used to detect total antioxidant capacity: Ascorbic acid standard solutions of different concentrations (0-30 μM), 100 μL TMB solution (0.1 mM), 100 μL ZE solution, and 700 μL acetate buffer (pH 4.0, 20 mM) were mixed and reacted under visible light for 7 minutes. The absorbance at 652 nm was then measured. Figure 6 As shown, the ascorbic acid concentration exhibited a linear relationship with absorbance changes within the range of 0-30 μM, with a detection limit of 0.18 μM. Spiking recovery experiments were conducted on commercially available fruit juice, yielding recoveries of 99.1%-102.5%, as shown in Table 1.

[0035] Table 1. Detection of AA in fruit juice

[0036] Dosage (μM) Measured value (μM) Recovery rate (%) Relative standard deviation (%, n=3) 0.00 2.92 ± 0.54 — — 5.00 7.88 ± 0.55 99.1 1.53 10.00 13.17 ± 0.22 102.5 1.92 20.00 19.64 ± 0.61 98.2 4.03

[0037] Peroxidase-like activity was used for glucose detection: glucose oxidase was incubated with glucose standard solutions of different concentrations (5-70 μM) for 30 minutes, ZE and TMB were added, and the reaction was carried out in the dark for 10 minutes. The absorbance at 652 nm was then measured. Figure 7 As shown, glucose concentration exhibited a linear relationship with absorbance in the range of 5-70 μM, with a detection limit of 2.7 μM. Spiking recovery experiments were conducted on commercially available fruit juice, yielding recoveries of 99.1-101.4%. The analytical results are shown in Table 2.

[0038] Table 2. Detection of glucose in fruit juice

[0039] Dosage (μM) Measured value (μM) Recovery rate (%) Relative standard deviation (%, n=3) 0.00 5.98±0.28 — — 10.00 15.89±0.32 99.1 1.03 30.00 36.47±0.75 101.4 2.06 50.00 56.10±0.19 100.2 0.34

[0040] In summary, the simulated enzyme provided in this embodiment of the invention can be used for colorimetric detection of total antioxidant capacity, and can also be used for colorimetric detection of glucose in a cascade reaction with glucose oxidase. It has the advantages of simple operation, rapid response, high sensitivity, good selectivity and low cost, and has good application prospects in food analysis, clinical diagnosis and other fields.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a zein-based mimic enzyme with light-controlled dual-enzyme activity, characterized in that, Includes the following steps: Step S1: Dissolve zein in an alkaline aqueous solution and incubate under water bath conditions; Step S2: Add hydroxylamine solution to the solution obtained in step S1 and continue the reaction; Step S3: After the reaction is complete, a zein-based mimic enzyme with light-controlled dual-enzyme activity is obtained.

2. The method for preparing the zein-based mimic enzyme with light-controlled dual-enzyme activity according to claim 1, characterized in that, In S1, the alkaline aqueous solution is a sodium hydroxide solution with a concentration of 0.01-0.1 M.

3. The method for preparing the zein-based mimic enzyme with light-controlled dual-enzyme activity according to claim 1, characterized in that, In S1, the temperature of the water bath is 50-70℃.

4. The method for preparing the zein-based mimic enzyme with light-controlled dual-enzyme activity according to claim 1, characterized in that, In S1, the concentration of zein after dissolving in an alkaline aqueous solution is 0.1-5 mg / mL.

5. The method for preparing the zein-based mimic enzyme with light-controlled dual-enzyme activity according to claim 1, characterized in that, In S2, the concentration of the hydroxylamine solution is 10-100 mM, and the reaction time is 10-30 minutes.

6. A zein-based mimic enzyme with light-controlled dual-enzyme activity, characterized in that, It is prepared using the preparation method described in any one of claims 1-5.

7. The application of a zein-based mimic enzyme with light-controlled dual-enzyme activity as described in claim 6 in colorimetric detection.

8. The application according to claim 7, characterized in that, The total antioxidant capacity was determined by using the zein-based mimic enzyme with light-controlled dual enzyme activity under visible light irradiation to measure its oxidase-like activity, or by using the peroxidase-like activity of the zein-based mimic enzyme with light-controlled dual enzyme activity under dark conditions to measure glucose.

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