Chiral quantum dot-based alpha-amylase activator, preparation method and application thereof

Abstract

The application discloses a kind of alpha-amylase activator based on chiral quantum dots and preparation method and application, including the following steps: using chiral amino acid L / D-penicillamine as chiral source, modified graphdiyne quantum dots (GDQDs), the surface functionalization of GDQDs is prepared chiral graphdiyne quantum dots L (D) -GDQDs;Alpha-amylase is fully dissolved in buffer, and is fully mixed with L (D) -GDQDs to prepare alpha-amylase activator.The application provides a kind of new method for preparing activator to improve the activity of alpha-amylase, wherein L (D) -GDQDs used have good water solubility, stability and biological activity, can be combined with alpha-amylase to change the structure of enzyme, so as to affect the activity of enzyme, and has a significant effect on improving the catalytic activity of enzyme.

Description

Technical Field

[0001] This invention belongs to the field of chemical engineering, specifically relating to an α-amylase activator based on chiral quantum dots, its preparation method, and its application. Background Technology

[0002] Chirality is an important biochemical property in biological systems, widely present in molecules, cells, and tissues. The ubiquity and importance of chiral molecules in organisms have led to widespread interest in the preparation and application of chiral materials in the biomedical field. The introduction of chirality can enhance cellular absorption and improve the therapeutic performance of nanomaterials. Functionalization of chiral nanomaterials with surface ligands can effectively reduce cytotoxicity, thus exhibiting good biocompatibility and applications in drug detection, biomedicine, and therapy. Studying the interaction between nanomaterials and chiral molecules is of significant importance to biology, drug processes, and pathology.

[0003] Due to the complexity of the cellular microenvironment, traditional nanomaterials face many challenges in practical applications due to their toxicity and other factors. In recent years, carbon nanomaterials have been increasingly recognized as one of the most promising classes of nanomaterials due to their low toxicity, chemical stability, and optical properties. Graphdiyne (GDY) possesses a large conjugated system, a natural and regular porous structure, good chemical catalytic performance, and chemical stability; therefore, GDY is considered an excellent support for improving bioactive catalysts. Graphdiyne quantum dots (GDQDs) are a novel carbon-based low-dimensional semiconductor material. Due to their small size and unique electronic structure, they exhibit good optical stability and biocompatibility, making them promising for applications in optoelectronic materials, biomedicine, and other fields. Compared with other carbon nanomaterials, GDQDs have more acetylene active units and more surface defects, thus exhibiting higher bioactivity. The hydrophobic interactions and π-π stacking between unsaturated bonds in GDQDs, as well as the abundant oxygen-containing functional groups on their surface, can act as excellent ligands to bind biomolecules and interact with enzymes.

[0004] Enzymes are highly specific biocatalysts that accelerate various biochemical reactions, ensuring the normal progress of life processes. However, due to their high cost and low stability, the application of enzyme treatment is currently limited. Exploring new methods for enzyme treatment aims to reduce enzyme-related costs and improve process efficiency. α-Amylase is an enzyme that catalyzes the formation of dextrin, glucose, maltose, and other molecules from starch molecules, and it plays an important role in biotechnology, pharmacy, food, detergents, glucose syrup, and beverage production. To date, research on α-amylase activators is scarce. α-Amylase is unstable to salt, high temperature, extreme pH, organic solvents, and toxic reagents. Enzyme performance can be improved and activity enhanced by binding the enzyme to a suitable carrier. The unique physicochemical properties of nanomaterials can improve enzyme loading and regulate the catalytic activity of industrially and clinically useful enzymes. However, current technologies mostly use organic synthetic polymers and metal nanoparticles to improve the treatment effect of α-amylase, which have certain biotoxicity and are not safe for use in the food industry and pharmaceutical fields. Currently, there are no research reports on using chiral quantum dots as carriers for α-amylase activators. Summary of the Invention

[0005] Purpose of the invention: To address the problems existing in the prior art, this invention provides a method for preparing an α-amylase activator based on chiral quantum dots. The activator prepared by this invention has good stability and biocompatibility, and significantly improves the catalytic activity of α-amylase.

[0006] The present invention also provides the prepared chiral quantum dot-based α-amylase activator and its applications.

[0007] Technical Solution: To achieve the above objectives, the present invention provides a method for preparing an α-amylase activator based on chiral quantum dots, comprising the following steps:

[0008] (1) Preparation of graphyne quantum dot solution: Disperse graphyne quantum dots GDQDs to obtain graphyne quantum dot solution;

[0009] (2) Preparation of α-amylase stock solution: Dissolve α-amylase powder fully in buffer solution, centrifuge and take the supernatant to obtain α-amylase stock solution;

[0010] (3) Preparation of chiral graphyne quantum dots: 1-ethyl-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and L-penicillamine or D-penicillamine (L-PEN or D-PEN) solution are added to the quantum dot solution obtained in step (1) and stirred thoroughly. After dialyzing, the dialysate is freeze-dried to obtain solid chiral graphyne quantum dots L-GDQDs or D-GDQDs.

[0011] (4) Preparation of α-amylase activator: Disperse the L-GDQDs or D-GDQDs obtained in step (3) to obtain a chiral quantum dot solution, and mix the enzyme stock solution obtained in step (2) with the chiral quantum dot solution in a water bath to obtain the enzyme activator.

[0012] In step (1), the graphyne quantum dots GDQDs are synthesized from graphdiyne by a hydrothermal method.

[0013] In step (1), the concentration of the graphdiyne quantum dot (GDQDs) solution is 1–5 mg / mL.

[0014] In step (2), the buffer solution is a 0.1-0.2 mol / L citrate-sodium citrate buffer solution with a pH of 5-6.

[0015] Preferably, the buffer solution in step (2) is a 0.1 mol / L citrate-sodium citrate buffer solution with a pH value of 5 to 6, and the preferred pH value is 5.6.

[0016] In step (3), the volume ratio of EDC solution to NHS solution is 3 to 4:1.

[0017] Preferably, the volume ratio of EDC solution to NHS solution in step (3) is 4:1.

[0018] In step (3), the L-GDQDs or D-GDQDs have a particle size of 2 to 6 nm.

[0019] In step (4), the concentration of the L-GDQDs or D-GDQDs chiral quantum dot solution is 1 to 5 mg / mL.

[0020] In step (4), the volume ratio of α-amylase stock solution to chiral quantum dot solution is 1 to 4:1, and the mixing time in a water bath at 40 to 50°C is 2 to 6 hours.

[0021] Preferably, the method for preparing the α-amylase activator based on chiral quantum dots of the present invention includes the following steps:

[0022] (1) Preparation of Graphyne quantum dot solution: Graphyne quantum dots GDQDs are dispersed in double-distilled water to obtain Graphyne quantum dot solution.

[0023] (2) Preparation of α-amylase stock solution: Homogenize α-amylase powder in buffer solution, shake and mix every 5 min, and then let stand to extract. Repeat this operation four times. After centrifugation, take the supernatant and add 0.1 mol / L citrate-sodium citrate buffer (pH 5.6) to dilute to 10 mL. Mix the solution thoroughly to obtain α-amylase stock solution.

[0024] (3) Preparation of chiral graphyne quantum dots: L-PEN (or D-PEN) was used as the chiral source, and GDQDs were activated using the EDC / NHS method. EDC solution was added to the GDQDs solution obtained in step (1), and after stirring for 10 min, NHS solution was added, and after stirring for 30 min, L-PEN (or D-PEN) solution was added, and the mixture was stirred thoroughly for 6–10 h. Excess amino acids were removed by dialysis using a dialysis bag (molecular weight cutoff 500 Da), and the dialysate was freeze-dried to obtain solid chiral graphyne quantum dots L-GDQDs (or D-GDQDs).

[0025] (4) Preparation of α-amylase activator: Dissolve the L-GDQDs (or D-GDQDs) obtained in step (3) in double-distilled water to obtain a chiral quantum dot solution. Mix the enzyme stock solution obtained in step (2) and the chiral quantum dot solution in a certain proportion in a 40°C water bath to obtain the enzyme activator.

[0026] The α-amylase activator based on chiral quantum dots prepared by the preparation method described in this invention.

[0027] The application of the chiral quantum dot-based α-amylase activator described in this invention in improving the catalytic activity of α-amylase.

[0028] Furthermore, using 1% starch as the enzymatic hydrolysis substrate, a quantitative enzyme activator was added, and DNS reagent was added as a colorimetric reagent. The absorbance of the product at 540 nm was measured using a UV-Vis spectrophotometer, and a blank sample control was prepared to determine the relative activity of the enzyme.

[0029] This invention proposes for the first time the application of chiral graphyne quantum dots in the regulation of α-amylase activity. The invention prepares chiral graphyne quantum dots (L / D-GDQDs) by coupling the chiral amino acid L / D-penicillamine (L / D-PEN) with graphyne quantum dots (GDQDs). The resulting L / D-GDQDs have abundant active groups on their surface, exhibiting chirality, good water solubility and stability, as well as good biological activity, high fluorescence intensity and photostability. Upon interaction with α-amylase, they can effectively enhance enzyme activity. This invention synthesizes chiral graphyne quantum dots L-GDQDs and D-GDQDs, and interacts them with α-amylase to achieve the regulation of α-amylase activity by chiral quantum dots. Furthermore, it proposes a novel activator and preparation method for enhancing α-amylase activity, effectively improving the catalytic activity of α-amylase.

[0030] The graphyne quantum dots (GDQDs) used in this invention were obtained by an ultrasonic hydrothermal method. Chiral graphyne quantum dots (L(D)-GDQDs) were prepared using a two-step method. A chiral amino acid (L / D-PEN) was used as the chiral source, and GDQDs were modified using an EDC / NHS activation method to synthesize chiral graphyne quantum dots (L(D)-GDQDs). The DNS colorimetric method confirmed that these chiral quantum dots significantly affected the activity of α-amylase; both L-GDQDs and D-GDQDs could effectively improve the activity of α-amylase.

[0031] The graphyne quantum dots (GDQDs) used in this invention are a novel carbon-based low-dimensional semiconductor material. They not only possess the excellent optical properties and ease of functionalization inherent in traditional carbon-based quantum dots, but also exhibit richer carbon chemical bonds, a larger π-electron conjugated system, and a larger specific surface area. The surface of GDQDs contains abundant oxygen-containing active groups, which provide powerful and multifunctional active sites. Amino acids react with these active groups to form amide bonds, functionalizing the nanoparticle surface and completing the coupling of chiral groups with GDQDs. The L(D)-GDQDs surface possesses naturally regular nanopores, more acetylene active units, and surface defects. Its hydrophobic and π-conjugated surface can load many functional molecules, allowing biomolecules to anchor on its surface as binding sites, thus exhibiting higher biological activity. This invention uses L / D-PEN as a chiral source to synthesize chiral graphyne quantum dots and, for the first time, discovers that L(D)-GDQDs can bind to α-amylase, altering the enzyme's structure and significantly improving its activity. L(D)-GDQDs can be used to regulate the activity of α-amylase, significantly improving enzyme catalytic performance and showing broad application prospects in industry and medicine. This invention proposes a novel method for preparing an α-amylase activator, utilizing chiral graphyne quantum dots to regulate enzyme activity, providing a new approach for the application of graphyne quantum dots in the biological field. This method is simple, easy to implement, and environmentally friendly, with broad application prospects in the food, feed, and pharmaceutical industries. Therefore, chiral graphyne quantum dots are of great significance as regulators of enzyme activity.

[0032] The chiral graphdiyne quantum dots prepared in this invention possess a larger specific surface area, suitable nanopore shape, and surface charge that facilitates cell penetration and enzyme binding. They also exhibit low biotoxicity, effectively addressing the instability and low activity of α-amylase under conditions such as high temperatures, extreme pH, organic solvents, and toxic reagents. This invention proposes a novel method for regulating α-amylase activity by utilizing chiral graphdiyne quantum dots, providing a new approach for the application of graphdiyne quantum dots in the biological field. This effectively improves upon current methods that often use organic synthetic polymers and metal nanoparticles to enhance α-amylase treatment, which suffer from certain biotoxicity and are unsafe for use in the food and pharmaceutical industries. The chiral graphdiyne quantum dots synthesized in this invention demonstrate significant treatment effects, reduce the use of heavy metals and organic reagents, exhibit low toxicity, and significantly improve the treatment effect and stability of α-amylase.

[0033] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0034] 1. The chiral GDQDs prepared by the method of the present invention are activated by ultrasonic hydrothermal method, which has a shorter preparation cycle, reduces the use of heavy metals and other organic reagents, simplifies chemical oxidation and dialysis steps, reduces costs and is more environmentally friendly.

[0035] 2. The chiral GDQDs prepared by the method of the present invention are small in size, have a large specific surface area, have a suitable nanopore shape and a surface charge that allows them to easily penetrate into cells and bind to enzymes. They can bind to enzymes and thus interact more fully, making them an effective biocatalyst carrier with more significant enzyme interaction effects.

[0036] 3. Based on the interaction between chiral graphdiyne quantum dots and enzymes, this invention applies the specially prepared chiral quantum dots to the regulation of α-amylase activity for the first time. Compared with pure enzymes, the prepared α-amylase activator has significantly improved activity and stability. Compared with other enzyme activity regulators, this α-amylase activator has low biotoxicity, good water solubility, stability and bioactivity, high fluorescence intensity and photostability, and has broad application prospects in the field of biology. Attached Figure Description

[0037] Figure 1 This is a transmission electron microscope image of the L-GDQDs prepared in this invention;

[0038] Figure 2 This is a transmission electron microscope image of the D-GDQDs prepared in this invention;

[0039] Figure 3 These are the toxicity analysis results of L(D)-GDQDs prepared in this invention on MCF-7 cells;

[0040] Figure 4 This is a comparison chart showing the effects of the activator prepared in this invention on α-amylase;

[0041] Figure 5 The relative activities of α-amylase under the action of the activator prepared in this invention at different pH values;

[0042] Figure 6 The values ​​represent the relative activities of α-amylase under the action of the activator prepared in this invention at different temperatures. Detailed Implementation

[0043] The present invention will be further described below with reference to the embodiments.

[0044] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0045] The graphdiyne GDY described in this invention was synthesized according to the literature (Architecture of graphdiyne nanoscale films. Guoxing Li, Yuliang Li, Huibiao Liu, Yanbing Guo, Yongjun Li, Daoben Zhu. Chem. Commun., 2010, 46, 3256-3258), and the prepared graphdiyne is graphidyne.

[0046] The L-PEN used in this invention was purchased from Aladdin, part number S161218. The D-PEN used in this invention was purchased from Aladdin, part number P115952.

[0047] The α-amylase powder used in this invention was purchased from Macklin, catalog number A800731.

[0048] Example 1

[0049] Preparation of L(D)-GDQDs and formulation of α-amylase stock solution

[0050] 1. Preparation of L(D)-GDQDs

[0051] (1) Preparation of GDQDs: 20 mL of graphyne GDY aqueous solution (1 mg / mL) was ultrasonically treated at 400 W for 2 h to obtain a small-sized GDY solution. 6 mL of the above GDY solution was transferred to a reaction vessel and hydrothermally heated at 180 °C for 5 h. After cooling to room temperature, the resulting solution was dialyzed in a 500 Da molecular weight cutoff dialysis bag for 1 day, with the water changed every 8 h. The dialysate was then freeze-dried to obtain GDQDs.

[0052] (2) Preparation of L(D)-GDQDs: The synthesized GDQDs were dispersed in double-distilled water to obtain a graphyne quantum dot solution (1 mg / mL). Chiral graphyne quantum dots were prepared using a two-step method, with L / D-PEN as the chiral source. GDQDs were activated using the EDC / NHS method. EDC solution (120 μL, 6 mmol / L) was added to 1 mL of the GDQDs solution, and the mixture was stirred for 10 min. Then, NHS solution (30 μL, 6 mmol / L) was added, and the mixture was stirred for 30 min. Finally, L-PEN (or D-PEN) solution (30 μL, 6 mmol / L) was added, and the mixture was slowly stirred for 8 h. All reactions were carried out at room temperature. Excess amino acids were removed by dialysis using a dialysis bag (molecular weight cutoff 500 Da). The dialysate was then freeze-dried to obtain solid chiral graphyne quantum dots L-GDQDs (or D-GDQDs). Figure 1 , Figure 2 L-GDQDs or D-GDQDs can be observed, with a particle size of 2–6 nm.

[0053] The cytotoxicity of the prepared L-GDQDs and D-GDQDs to cells was evaluated using the MTT assay, such as... Figure 3 As shown in the figure, MCF-7 cells were co-incubated with different concentrations (0-1200 μg / mL) of L-GDQDs or D-GDQDs at 37°C for 24 h. The cell viability of MCF-7 cells was then detected using the MTT assay kit. The results showed that the cell viability remained above 90% after 24 h of interaction with different concentrations of L-GDQDs or D-GDQDs quantum dots, indicating that these chiral GDQDs have low toxicity and good biocompatibility.

[0054] 2. Preparation of α-amylase stock solution: Mix 0.1 g of α-amylase powder thoroughly in 1 mL of 0.1 mol / L citrate-sodium citrate buffer (pH 5.6). Shake well every 5 min, then allow to stand for extraction. Repeat this process four times. Centrifuge at 8000 rpm for 10 min at room temperature. Take all the supernatant and bring the volume to 10 mL with 0.1 mol / L citrate-sodium citrate buffer. Mix thoroughly to obtain the α-amylase stock solution.

[0055] Example 2

[0056] The L-GDQDs prepared in Example 1 were dispersed in double-distilled water to obtain an L-GDQDs solution (1 mg / mL). 500 μL of this quantum dot solution and 500 μL of α-amylase stock solution (prepared in Example 1) were added to 1 mL of 0.1 mol / L citrate-sodium citrate buffer (pH 5.6) and incubated in a water bath at 40°C for 3 h to obtain the enzyme activator.

[0057] Take 2 mL of enzyme activator and incubate it in a 40°C water bath. Add 1.0 mL of 1% starch solution (pre-incubated in a 40°C water bath), then add 1.0 mL of DNS reagent and boil in a water bath for 5 minutes. The effect is as follows. Figure 4 As shown, the absorbance of the product at 540 nm was measured using a UV-Vis spectrophotometer. An α-amylase solution of the same concentration was used as a blank control, and tests were conducted under the same conditions. The maltose content was calculated from the maltose standard curve using the obtained absorbance values. With the untreated pure enzyme activity as 100%, the relative activity under the action of the enzyme activator was calculated to be 143.18% of that of the pure enzyme using a formula.

[0058]

[0059] Among them, sample c and control c represent the content of maltose, a product of the enzyme activator and α-amylase reaction, respectively.

[0060] Example 3

[0061] The D-GDQDs prepared in Example 1 were dispersed in double-distilled water to obtain a D-GDQDs solution (1 mg / mL). 500 μL of this quantum dot solution and 500 μL of α-amylase stock solution (prepared in Example 1) were added to 1 mL of 0.1 mol / L citrate-sodium citrate buffer (pH 5.6), and the solution was incubated at 40°C for 3 h to obtain the enzyme activator. 2 mL of the enzyme activator was placed in a 40°C water bath and incubated. 1.0 mL of a 1% starch solution (pre-incubated at 40°C) was added, followed by 1.0 mL of DNS reagent and a boiling water bath for 5 min. The absorbance of the product at 540 nm was measured using a UV-Vis spectrophotometer. An α-amylase solution of the same concentration was used as a blank control, and tests were conducted under the same conditions. The maltose content was calculated from the absorbance using a maltose standard curve, and the relative enzyme activity was found to be 139.29% of the pure enzyme activity.

[0062] Example 4

[0063] Weigh 5g of germinated wheat seeds, place them in a mortar, add 10mL of double-distilled water, grind until homogeneous, and then shake to mix every 5 minutes. Repeat this process four times. Centrifuge at 8000r at room temperature for 10 minutes, and collect all the supernatant. Dilute to 10mL with 0.1mol / L citrate-sodium citrate buffer (pH 5.6), and mix thoroughly to obtain the enzyme extract. Disperse the L-GDQDs prepared in Example 1 in double-distilled water to obtain an L-GDQDs solution (1mg / mL). Take 500μL of this quantum dot solution and 500μL of the enzyme extract and add them to 1mL of 0.1mol / L citrate-sodium citrate buffer. Incubate at 40℃ for 3 hours to obtain the enzyme activator.

[0064] To eliminate the interference of β-amylase in germinating wheat seeds on the test results, the enzyme activator was heated in a 70℃ water bath for 15 min to inactivate the β-amylase. After cooling to room temperature, it was incubated in a 40℃ water bath. 1.0 mL of a 1% starch solution (pre-incubated in a 40℃ water bath) was added, followed by 1.0 mL of DNS reagent and a boiling water bath for 5 min. The absorbance of the product at 540 nm was measured using a UV-Vis spectrophotometer. An enzyme extract solution of the same concentration was used as a blank control, and tests were conducted under the same conditions. The maltose content was calculated from the absorbance using a maltose standard curve, and the relative enzyme activity was found to be 127.82% of that of the enzyme extract.

[0065] Example 5

[0066] The D-GDQDs prepared in Example 1 were dispersed in double-distilled water to obtain a D-GDQDs solution (1 mg / mL). 500 μL of this quantum dot solution and 500 μL of the enzyme extract prepared in Example 4 were placed in 1 mL of 0.1 mol / L citrate-sodium citrate buffer (pH 5.6) and incubated in a water bath at 40°C for 3 h to obtain the enzyme activator.

[0067] To eliminate the interference of β-amylase in germinating wheat seeds on the test results, 2 mL of enzyme activator was heated in a 70℃ water bath for 15 min to inactivate the β-amylase. After cooling to room temperature, it was incubated in a 40℃ water bath. 1.0 mL of 1% starch solution (pre-incubated in a 40℃ water bath) was added, followed by 1.0 mL of DNS reagent and a boiling water bath for 5 min. The absorbance of the product at 540 nm was measured using a UV-Vis spectrophotometer. An enzyme extract solution of the same concentration was used as a blank control and tested under the same conditions. The maltose content was calculated from the absorbance value using the maltose standard curve, and the relative enzyme activity was found to be 122.63% of that of the enzyme extract.

[0068] Comparative Example 1

[0069] The GDQDs prepared in Example 1 were dispersed in double-distilled water to obtain a GDQDs solution (1 mg / mL). 500 μL of this quantum dot solution and 500 μL of α-amylase stock solution (prepared in Example 1) were added to 1 mL of 0.1 mol / L citrate-sodium citrate buffer (pH 5.6) and incubated in a water bath at 40°C for 3 h to obtain an enzyme treatment solution.

[0070] The enzyme treatment solution was incubated in a 40°C water bath. 1.0 mL of a 1% (w / w) starch solution (pre-incubated in a 40°C water bath) was added, followed by 1.0 mL of DNS reagent and a boiling water bath for 5 min. The absorbance of the product at 540 nm was measured using a UV-Vis spectrophotometer. An α-amylase solution of the same concentration was used as a blank control, and tests were conducted under the same conditions. The maltose content was calculated from the absorbance using a maltose standard curve, revealing that the relative enzyme activity was 109.51% of the pure enzyme activity.

[0071] Comparative Example 2

[0072] The GDQDs prepared in Example 1 were dispersed in double-distilled water to obtain a GDQDs solution (1 mg / mL). 500 μL of this quantum dot solution and 500 μL of the enzyme extract prepared in Example 4 were added to 1 mL of 0.1 mol / L citrate-sodium citrate buffer (pH 5.6), and the solution was incubated at 40°C for 3 h to obtain the enzyme treatment solution. The enzyme treatment solution was heated in a 70°C water bath for 15 min, cooled to room temperature, and then incubated in a 40°C water bath. 1.0 mL of a 1% starch solution (pre-incubated at 40°C) was added, followed by 1.0 mL of DNS reagent, and then the solution was boiled for 5 min. The absorbance of the product at 540 nm was measured using a UV-Vis spectrophotometer. An enzyme extract solution of the same concentration was used as a blank control, and tests were conducted under the same conditions. The maltose content was calculated from the absorbance using a maltose standard curve, and the relative enzyme activity was found to be 102.37% of the pure enzyme activity.

[0073] Comparative Example 3

[0074] Take 2 mL of the enzyme activator prepared in Examples 2 and 3 respectively, adjust the pH of the enzyme activator to 3.6 using HCl-NaOH, and incubate both enzyme activators in a 40°C water bath. Add 1.0 mL of 1% starch solution (pre-incubated in a 40°C water bath) to each, and add 1.0 mL of DNS reagent to each, then boil in a water bath for 5 min. Measure the absorbance of the product at 540 nm using a UV-Vis spectrophotometer. Use an α-amylase solution of the same concentration as a blank control group and test under the same conditions. Calculate the maltose content from the absorbance values ​​using the maltose standard curve. Repeat this experiment by changing the pH value to obtain the relative enzyme activity at different pH values, such as... Figure 5 As shown, under the action of the enzyme activator, the relative activity of the enzyme is increased to varying degrees at different pH values. The enzyme activator based on chiral GDQDs prepared in this invention significantly enhances the acid resistance and stability of the enzyme.

[0075] Comparative Example 4

[0076] Take 2 mL of the enzyme activator prepared in Examples 2 and 3 respectively, and incubate both enzyme activators in a 30°C water bath. Add 1.0 mL of 1% starch solution (pre-incubated in a 30°C water bath) to each, followed by 1.0 mL of DNS reagent and boiling water bath for 5 min. Measure the absorbance of the product at 540 nm using a UV-Vis spectrophotometer. Use an α-amylase solution of the same concentration as a blank control group and test under the same conditions. Calculate the maltose content from the absorbance values ​​using the maltose standard curve. Repeat this experiment by changing the reaction temperature to obtain the relative enzyme activity at different temperatures, such as... Figure 6 As shown, under the action of enzyme activator, the relative activity of enzymes is increased to varying degrees at different temperatures. The enzyme activator based on chiral GDQDs prepared in this invention significantly enhances the enzyme's heat resistance and stability.

[0077] The enzyme catalytic activity of the enzyme activator based on chiral GDQDs prepared in this invention is significantly improved, and the catalytic effect is better than that of GDQDs themselves, while exhibiting low toxicity and good stability. Therefore, this invention can be applied to enhance the activity of α-amylase and, as a novel enzyme activity regulator, has broad application prospects in industrial and biomedical fields.

Claims

1. A method for preparing an α-amylase activator based on chiral quantum dots, characterized in that, Includes the following steps: (1) Preparation of graphyne quantum dot solution: Graphyne quantum dots GDQDs were dispersed to obtain graphyne quantum dot solution; (2) Preparation of α-amylase stock solution: Dissolve α-amylase powder fully in buffer solution, centrifuge and take the supernatant to obtain α-amylase stock solution; (3) Preparation of chiral graphyne quantum dots: 1-ethyl-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and L-penicillamine or D-penicillamine (L-PEN or D-PEN) solution are added to the quantum dot solution obtained in step (1) and stirred thoroughly. After dialyzing, the dialysate is freeze-dried to obtain solid chiral graphyne quantum dots L-GDQDs or D-GDQDs. (4) Preparation of α-amylase activator: Disperse the L-GDQDs or D-GDQDs obtained in step (3) to obtain a chiral quantum dot solution, and mix the enzyme stock solution obtained in step (2) with the chiral quantum dot solution in a water bath to obtain the enzyme activator.

2. The preparation method according to claim 1, characterized in that, In step (1), the graphyne quantum dots GDQDs are synthesized from graphdiyne via a hydrothermal method.

3. The preparation method according to claim 1, characterized in that, The concentration of the graphyne quantum dot (GDQDs) solution in step (1) is 1–5 mg / mL.

4. The preparation method according to claim 1, characterized in that, In step (2), the buffer solution is preferably a 0.1-0.2 mol / L citrate-sodium citrate buffer solution with a pH value of 5-6.

5. The preparation method according to claim 1, characterized in that, In step (3), the volume ratio of EDC solution to NHS solution is 3 to 4:

1.

6. The method according to claim 1, characterized in that, The L-GDQDs or D-GDQDs in step (3) have a particle size of 2 to 6 nm.

7. The method according to claim 1, characterized in that, In step (4), the concentration of the L-GDQDs or D-GDQDs chiral quantum dot solution is 1 to 5 mg / mL.

8. The method according to claim 1, characterized in that, In step (4), the α-amylase stock solution and the chiral quantum dot solution are mixed in a volume ratio of 1 to 4:1 in a water bath at 40 to 50°C for 2 to 6 hours.

9. An α-amylase activator based on chiral quantum dots prepared by the preparation method of claim 1.

10. The application of the chiral quantum dot-based α-amylase activator of claim 9 in improving the catalytic activity of α-amylase.

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