Application of fullerene derivative in protecting and / or improving enzyme activity

Abstract

The present disclosure relates to protection and / or enhancement of enzymatic activity. The invention provides an application of a fullerene derivative in protecting and / or improving enzyme activity, the fullerene derivative has a modification group for increasing water solubility, and an enzyme protective agent comprising the fullerene derivative. The fullerene derivative material disclosed by the invention is stable in structure, good in water solubility and excellent in biocompatibility, and can effectively remove damage factors around an enzyme under the condition which is possibly encountered in industrial production and enzyme catalysis engineering and is not beneficial to enzyme catalysis activity, so that the efficiency of a catalytic substrate is improved, and the yield of the product is improved. A novel efficient enzyme protection material with excellent biocompatibility is constructed. The fullerene derivative material disclosed by the invention is non-toxic and non-dangerous, can be carried out under mild reaction conditions, is simple and controllable, is high in practicability, and effectively widens the variety range of protective enzymes.

Description

Technical Field

[0001] The present disclosure relates to the protection and / or improvement of enzyme activity, and in particular to the use of fullerene derivatives in the protection and / or improvement of enzyme activity. Background Art

[0002] In recent years, with the rapid development of the economy and the rapid progress of science and technology, enzymes have shown great superiority in the fields of organic synthesis, pharmaceuticals, food industry and environmental protection. Its advantages include high selectivity, efficient catalysis, mild reaction conditions, high tolerance for complex substrates, renewability and environmental friendliness. Enzymes are a kind of protein catalyst in organisms that can catalyze reactions and control complex metabolic processes with an efficiency that chemical catalysts cannot match. As green catalysts, enzymes also conform to the development trend of safe, healthy and environmentally friendly industrial production. Although enzymes have extremely high catalytic efficiency, the action conditions of natural enzymes are relatively harsh. Adverse conditions such as extreme pH and temperature, non-aqueous solvents, free radicals, and non-natural substrates will have a great impact on the catalytic effect of enzymes. In severe cases, it will cause changes in protein structure and inactivation. Therefore, the application of enzymes in non-cellular environments is limited.

[0003] Fullerene is an allotrope of carbon. This type of substance refers to a cage-like structure composed of carbon atoms, among which C 60 , C 70 Such materials have high symmetry and large conjugated electronic structures, which enable them to react efficiently with free radicals and neutralize their activity. Fullerenes can stabilize free radicals by capturing unpaired electrons on them, preventing them from initiating further oxidation reactions, and are known as "free radical sponges". At the same time, fullerenes have high stability, strong antioxidant properties, and good biocompatibility, making them an ideal bio-nanomaterial. Summary of the invention

[0004] The object of the present disclosure is to provide a method for protecting and / or improving enzyme activity, especially a method for protecting and / or improving enzyme activity under adverse conditions.

[0005] Another object of the present disclosure is to provide an enzyme protecting agent that can protect and / or improve enzyme activity, especially an enzyme protecting agent that can protect and / or improve enzyme activity under adverse conditions.

[0006] In order to solve the above technical problems and achieve the above objectives, the present disclosure provides the following technical solutions:

[0007] In a first aspect, the present disclosure provides the use of a compound represented by formula (I) in protecting and / or improving enzyme activity.

[0008]

[0009] Among them, in formula (I), F is fullerene, and the fullerene is selected from at least one of hollow fullerene, metal fullerene, heterocyclic fullerene, and endohedral fullerene;

[0010] R is a modifying group of the fullerene, and the modifying group is a modifying group for increasing water solubility;

[0011] m is the number of modifying groups, and 1 ≤ m ≤ 30.

[0012] In a second aspect, the present disclosure provides an enzyme protectant, which comprises the compound represented by formula (I).

[0013] In a third aspect, the present disclosure provides a drug, food, health product, and / or cosmetic comprising the compound represented by formula (I), which further comprises an enzyme.

[0014] In one embodiment of the above application, enzyme protectant, drug, food, health product, and / or cosmetic, the compound protects and / or enhances enzyme activity under adverse conditions;

[0015] Preferably, the adverse conditions include temperature, light, pH value, and / or free radical concentration that are not conducive to enzyme activity;

[0016] More preferably, the adverse conditions include light, pH value, and / or free radical concentration that are not conducive to enzyme activity;

[0017] Further preferably, the adverse conditions include a light duration of 1 - 12 hours, a pH value of 3 - 10, and / or a free radical concentration range of 0.25 - 2 mM.

[0018] In another embodiment of the above application, enzyme protectant, drug, food, health product, and / or cosmetic, one end of R is combined with the fullerene through an alkyl chain, a nitrogen-containing group, a phenyl group, or a mercapto group, and the other end of R is a group containing a carboxyl group, an amino group, and / or a hydroxyl group.

[0019] Preferably, when the other end of R is a hydroxyl group, 1 ≤ m ≤ 4 or 15 < m < 30. For example, the number of modifying groups of hydroxyl in the fullerene derivative hydroxyfullerene (THPC) can be 4. For example, the number of modifying groups of hydroxyl in the fullerene derivative C 60 -OH or C 70 -OH can be controlled to be 15 - 30 by adjusting the reaction temperature and reaction time during its preparation process (CN107556166A).

[0020] Preferably, when the other end of R is a group other than a hydroxyl group, 1 ≤ m < 4.

[0021] In another embodiment of the above application, enzyme protectant, drug, food, health product, and / or cosmetic, the fullerene is selected from hollow fullerene C2a , at least one of metal fullerene A2C2@C 2b or metal fullerene B3N@C2b, wherein 30 < a < 50, 39 ≤ b ≤ 44, A is at least one of Sc, La, Y; B is at least one of Sc, La, Y, Ho, Lu, Er;

[0022] Preferably, a is 30, 35, 38, 39, 41 or 42;

[0023] Preferably, b is 41 or 42.

[0024] In another embodiment of the above application, enzyme protectant, drug, food, health product and / or cosmetic, the compound is selected from C 70 -OH, C 60 -OH, amino fullerene (TAPC), carboxyl fullerene (TCPC), hydroxyl fullerene (THPC), C 60 (C(COOH)2)2, C 70 (C(COOH)2)2, Sc3N@C80(C(COOH)2)2, hyaluronic acid modified fullerene nanomaterial (HA-C 60 ), cyclodextrin modified fullerene nanomaterial, polyethylene glycol modified fullerene nanomaterial (PEG-C 60 ), polyvinylpyrrolidone modified fullerene nanomaterial (PVP-C 60 ), polyvinyl ketone modified fullerene nanomaterial (PVA-C 60 ) at least one.

[0025] In another embodiment of the above application, enzyme protectant, drug, food, health product and / or cosmetic, the concentration of the compound is 0.2 - 2 mg / mL; preferably, the concentration of the compound is 0.8 - 1.5 mg / mL; more preferably, the concentration of the compound is 0.8 mg / mL.

[0026] In another embodiment of the above application, enzyme protectant, drug, food, health product and / or cosmetic, the enzyme is selected from at least one of glucose-6-phosphate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, α-ketoglutarate dehydrogenase, enolpyruvate dehydrogenase and formate dehydrogenase;

[0027] Preferably, the enzyme is alcohol dehydrogenase, aldehyde dehydrogenase and / or formate dehydrogenase;

[0028] Preferably, when the enzyme is alcohol dehydrogenase, the general formula of its substrate is S-OH, where S is selected from -CH3, -C2H5, -C4H9, -C5H 11, -CH(CH3)2, -CH2CH(CH3)2, -CH2C6H5 and -CH2CH2OH; More preferably, S is at least one selected from -CH3, -C2H5 and -CH2C6H5;

[0029] Preferably, when the enzyme is acetaldehyde dehydrogenase, its substrate is CH3CHO and / or C2H5CHO; more preferably, the substrate is CH3CHO:

[0030] Preferably, when the enzyme is formate dehydrogenase, its substrate is HCOOH.

[0031] The beneficial effects achieved by the present disclosure include:

[0032] The present disclosure breaks through the technical problems of poor biocompatibility, poor water solubility, and poor stability of traditional enzyme protection materials, and uses fullerene derivatives to improve the activity of enzymes, accelerate the rate of enzyme catalytic reactions, and thus extend the life of enzymes. The fullerene derivative materials disclosed in the present disclosure have stable structures, good water solubility, and excellent biocompatibility. Under conditions that are not conducive to enzyme catalytic activity that may be encountered in industrial production and enzyme catalytic engineering, they can effectively remove destructive factors around the enzyme, thereby improving the efficiency of catalytic substrates, and constructing a new type of high-efficiency enzyme protection material with excellent biocompatibility. The fullerene derivative materials disclosed in the present disclosure are non-toxic, non-hazardous, can be carried out under mild reaction conditions, are simple and controllable, have strong practicality, and effectively broaden the range of enzyme protection types. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the structural formula and characteristic absorption peak of cofactor NAD+ and product NADH in dehydrogenase activity detection.

[0034] Figure 2 This is the concentration-peak area standard curve of the reaction between acetaldehyde and 2,4-dinitrophenylhydrazone in the detection of alcohol dehydrogenase activity.

[0035] Figure 3 For no C 70 Ultraviolet absorption spectrum of the reaction solution after half an hour of alcohol dehydrogenase-catalyzed reaction treated with light for 3 hours in the presence of -OH.

[0036] Figure 4 For C 70 Ultraviolet absorption spectrum of the reaction solution after half an hour of alcohol dehydrogenase-catalyzed reaction treated with light for 3 hours in the presence of -OH.

[0037] Figure 5 Shows C 70 -OH effect on the activity of alcohol dehydrogenase under light damage conditions. The left figure shows the absence of C 70UV absorption spectrum and HPLC peak area of ​​the reaction solution after 24 hours of alcohol dehydrogenase catalytic reaction after 3 hours of light treatment in the presence of -OH. The right figure shows the peak area of ​​alcohol dehydrogenase catalytic reaction in the presence of C 70 Ultraviolet absorption spectrum of the reaction solution and HPLC detection peak area after half an hour of alcohol dehydrogenase-catalyzed reaction treated with light for 3 hours in the presence of -OH.

[0038] Figure 6 The results show that C 70 -OH on the activity of alcohol dehydrogenase, where - is the control group (no C 70 -OH), + is the experimental group (with C 70 -OH), and the hydrogen peroxide concentration of the blank group was 0.

[0039] Figure 7 Shows C 70 Effect of -OH on the activity of alcohol dehydrogenase under different pH conditions, where - is the control group (no C 70 -OH), + is the experimental group (with C 70 -OH).

[0040] Figure 8 Shows C 60 Effect of -OH on acetaldehyde dehydrogenase activity under different pH conditions, where - is the control group (no C 70 -OH), + is the experimental group (with C 70 -OH). DETAILED DESCRIPTION

[0041] (I) Definition

[0042] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0043] As used herein, the terms "a" and "an" and "the" and similar referents refer to both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0044] As used herein, the conjunction term "and / or" between various elements is intended to include both the meanings of "and" and "or", for example, the phrase "A, B and / or C" is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0045] As used herein, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any stated features, elements, integers, steps, or components, but not to preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of.

[0046] Numerical ranges used herein should be understood to include all numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0047] As used herein, the term "about" means a range of ±20% of the value that follows. In some embodiments, the term "about" means a range of ±10% of the value that follows. In some embodiments, the term "about" means a range of ±5% of the value that follows.

[0048] As used herein, the term "fullerene" is a series of spherical cluster molecules composed of an even number of carbon atoms, 12 five-membered rings, and the rest are six-membered rings, which are cage-like structures composed of carbon atoms. Fullerenes include hollow fullerenes, metallofullerenes, endohedral fullerenes, and heterocyclic fullerenes, and the hollow fullerene is a cage-like structure composed of carbon atoms alone.

[0049] As used herein, the term "endohedral fullerene" refers to a fullerene having one or more atoms embedded in a carbon cage, that is, a special type of molecule in which metal ions, metal-containing ion clusters, non-metallic atoms, molecules, etc. are embedded in a fullerene carbon cage. Endohedral fullerene not only has the physical and chemical properties of the fullerene carbon cage, but also has the characteristics of its embedded atoms or clusters.

[0050] As used herein, the term "metallofullerene" refers to an endohedral fullerene having metal atoms or atomic clusters embedded in a carbon cage, generally represented by the form of M@C2n, where M represents a metal element. 2b or metallofullerene B3N@C 2b At least one of the following, wherein: 39≤b≤44, optionally 41 or 42; A is at least one of Sc, La, and Y; B is at least one of Sc, La, Y, Ho, Lu, and Er.

[0051] As used herein, the term "heterocyclic fullerene" refers to a fullerene containing one or more heteroatoms other than carbon (such as nitrogen, boron, oxygen, sulfur, etc.) in its molecular structure, which replaces some of the carbon atoms in the fullerene molecule, giving the molecule new chemical and physical properties. Due to the introduction of heteroatoms, heterocyclic fullerenes are different from pure carbon fullerenes in terms of electronic properties, reactivity, etc., and therefore have potential application value in the fields of materials science, medicinal chemistry, nanotechnology, etc.

[0052] As used herein, the term "enzyme", also known as a biocatalyst, is a protein or RNA produced by living cells that has high specificity and high catalytic efficiency for its substrate.

[0053] As used herein, the terms "adverse conditions" and "destructive conditions" are used interchangeably and refer to factors that may affect the spatial structure of the enzyme during the catalytic process of the enzyme, ultimately causing the enzyme activity to decrease or even be lost.

[0054] As used herein, the term "protection" refers to a method of reducing or eliminating factors that may adversely affect the activity of an enzyme during the catalytic action of the enzyme.

[0055] (II) Detailed technical solution

[0056] Some embodiments of the present disclosure are described in detail below in conjunction with the accompanying drawings. In the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. The following embodiments are used to further illustrate the present disclosure, but should not be construed as limiting the present disclosure. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present disclosure should be equivalent replacement methods and are included in the protection scope of the present disclosure.

[0057] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0058] Example 1. Protective effect of C70-OH on alcohol dehydrogenase activity under adverse light conditions

[0059] (1) Effect of illumination time on enzyme activity

[0060] 45 mL of PBS solution of catalytic substrate ethanol (containing 9 mL of 50% ethanol aqueous solution and 36 mL of PBS solution) was evenly dispersed and then 5 mL of ethanol dehydrogenase with an initial enzyme activity of 50 U / mL was added. The air in the system was purged by N2 for 30 min, and the system was irradiated with a visible light source of λ>400 nm (xenon lamp light source equipped with a 420 nm filter) for 0.5, 1, 1.5, 2, 2.5, and 3 hours, respectively. Three replicates were set for each treatment group, and 15 mL of cofactor NAD was added. + After half an hour, 2 mL of the reaction solution was taken and the enzyme activity was detected in vitro without tissue using the Valle & Hoch method: Enzyme catalysis requires the cofactor NAD + After the catalysis is completed, NADH will be generated. This substance has a characteristic absorption peak at 340nm. The amount of NADH characteristic peak generated by UV-Vis detection reflects the activity of the enzyme. Figure 1 NAD + From the structural formula of NADH, we can know that NADH has characteristic absorption at 340nm. The value of this absorption peak can be used to judge the degree of reaction. When the reaction is complete, A 340 =0.88. The absorbance of NADH was measured by spectrophotometer and the enzyme activity was compared. The results are shown in Table 1.

[0061] As can be seen from Table 1, the longer the illumination time, the greater the damage to the enzyme. When the illumination time is less than or equal to 3 hours, the enzyme is not inactivated and can still complete catalysis after being left overnight. When the illumination time is greater than 3 hours, the enzyme is almost inactivated. 70 -OH plays a role in protecting enzyme activity under adverse conditions, and the illumination time was selected as 3 h in subsequent experiments.

[0062] Table 1. Effects of different illumination times on enzyme activity

[0063] Lighting time (h) <![CDATA[Absorbance A 340 > <![CDATA[After standing overnight, A 340 > 0.5 0.57 0.88 1 0.35 0.88 1.5 0.27 0.88 2 0.21 0.88 2.5 0.16 0.88 3 0.13 0.88 3.5 0.08 0.35

[0064] (2) Alcohol dehydrogenase initial enzyme activity test

[0065] 45 mL of PBS solution of catalytic substrate ethanol (containing 9 mL of 50% ethanol aqueous solution and 36 mL of PBS solution) was evenly dispersed and then 5 mL of alcohol dehydrogenase with different initial activities was added. The air in the system was purged by N2 for 30 min, and the system was irradiated with a visible light source of λ>400 nm (xenon lamp light source equipped with a 420 nm filter) for 3 hours. 15 mL of cofactor NAD was added. + After half an hour, 2 mL of the reaction solution was taken into the spectrophotometer to measure the absorbance of NADH and compare the enzyme activity. Three replicates were set for each treatment group, and the results are shown in Table 2.

[0066] As can be seen from Table 2, when the initial activity of alcohol dehydrogenase is 50-70 U / mL, the catalytic reaction can be completely carried out after being placed overnight. For cost saving considerations, the initial enzyme activity of 50 U / mL was selected in subsequent experiments.

[0067] Table 2. Catalytic performance test of different initial enzyme activities under 3 hours of light

[0068]

[0069] (3)C 70 Effect of -OH on the activity of alcohol dehydrogenase

[0070] 45 mL of PBS solution of catalytic substrate ethanol (containing 9 mL of 50% ethanol aqueous solution and 36 mL of PBS solution) was evenly dispersed and then 5 mL of ethanol dehydrogenase with an initial enzyme activity of 50 U / mL was added. N2 was passed through the system for 30 min to exhaust the air. After N2 was passed through the experimental group, 0.5 mL of C with a concentration of 0.8 mg / mL was added. 70 -OH, control group without C 70 -OH was replaced with PBS, and 3 replicates were set for each treatment. Irradiate with a visible light source with λ>400nm (xenon light source equipped with a 420nm filter) for 3 hours, and add 15mL of cofactor NAD + After half an hour, take 2 mL of the reaction solution and put it into the spectrophotometer to measure the absorbance of NADH. At the same time, take 2 mL of the reaction solution and mix it with 0.3 mL of 2,4-dinitrophenylhydrazine, adjust the pH to 2-4, and react for half an hour in the dark. Then, use HPLC to detect the product content to reflect the activity of alcohol dehydrogenase. The detection principle is: aldehyde and 2,4-dinitrophenylhydrazine can undergo nucleophilic addition reaction in an acidic environment to generate a red 2,4-dinitrophenylhydrazone solution, which is directly detected by high performance liquid chromatography-ultraviolet detector and quantified by external standard working curve method, which can accurately, quickly and stably detect the acetaldehyde content. Figure 2 For the concentration-peak area standard curve of acetaldehyde-2,4-dinitrophenylhydrazone, different concentrations of standard samples were prepared, and the peak area S corresponding to the peak time of 7.6 min in HPLC was detected to obtain the standard curve y=10 7 x+51046(R 2 =0.998). When the reaction was complete, S = 118,8046. During the experiment, HPLC used a ZORBAXSB-C18 column (4.6×150 mm×5 μm), and the mobile phase was 0.1% formic acid acetonitrile and 0.1% formic acid aqueous solution.

[0071] Figure 3 The test results of the control group show that 70 When -OH is present, the system is irradiated with light for 3 hours before adding the cofactor NAD+ After the light source was removed and the reaction lasted for half an hour, the absorbance of the characteristic absorption peak at 340nm was A=0.13, and S in HPLC was 37,4118. The reaction was almost not carried out, which indicated that the light conditions would destroy the activity of alcohol dehydrogenase. + After the light source was removed and the mixture was left for 24 hours, the activity of alcohol dehydrogenase was tested again by spectrophotometer and HPLC. Figure 5 As shown in the left figure, it can be found that the peak area of ​​HPLC is close to the data when the reaction is complete, which indicates that 3 hours of illumination did not inactivate the enzyme, but reduced the activity of the enzyme.

[0072] Figure 4 and Figure 5 The right picture shows the test results of the experimental group. It can be seen that when C is added into the system during illumination, 70 -OH, continued irradiation for 3 hours before adding cofactor NAD + After the light source was removed and the reaction lasted for half an hour, the absorbance of the characteristic absorption peak at 340nm was measured by UV spectrophotometer and was A=0.85. S=118,7582 was calculated in HPLC. These two data mutually verified that the enzyme catalyzed reaction in C 70 -OH can be protected normally, C 70 -OH protects alcohol dehydrogenase under light damage conditions and increases the reaction rate catalyzed by the enzyme.

[0073] (4) Different concentrations of C 70 Effect of -OH on the activity of alcohol dehydrogenase

[0074] 45 mL of PBS solution of catalytic substrate ethanol (containing 9 mL of 50% ethanol aqueous solution and 36 mL of PBS solution) was evenly dispersed and then 5 mL of ethanol dehydrogenase with an initial enzyme activity of 50 U / mL was added. N2 was passed through the system for 30 min to remove the air. 0.5 mL of C 70 -OH, irradiate with a visible light source of λ>400nm for 3 hours, add 15mL of cofactor NAD + After half an hour, 2 mL of the reaction solution was taken into the spectrophotometer to measure the absorbance of NADH and compare the enzyme activity. Three replicates were set for each treatment group, and the results are shown in Table 3.

[0075] Table 3. Different concentrations of C 70 -OH protection enzyme performance test under 3 hours of light

[0076] <![CDATA[C 70 -OH(mg / mL)]]> <![CDATA[Absorbance A 340 > 0.2 0.26 0.4 0.33 0.6 0.52 0.8 0.88 1 0.88 1.5 0.88 2 0.88

[0077] It can be seen from Table 3 that different concentrations of C 70-OH can protect the enzyme activity to a certain extent, and the material with good enzyme protection performance can be obtained at a concentration of 0.8-2 mg / mL, among which 0.8 mg / mL material concentration is the preferred condition.

[0078] Example 2. Protective effects of different fullerene derivative materials on enzymes under adverse light conditions

[0079] 45 mL of PBS solution of catalytic substrate ethanol (containing 9 mL of 50% ethanol aqueous solution and 36 mL of PBS solution) was evenly dispersed and then 5 mL of ethanol dehydrogenase with an initial enzyme activity of 50 U / mL was added. N2 was passed through the system for 30 minutes to exhaust the air. After passing N2, 0.5 mL of different types of fullerene derivatives with a concentration of 0.8 mg / mL were added to the experimental group. The control group had no fullerene derivatives and was replaced with PBS solution. Three replicates were set for each group. Irradiate with a visible light source of λ>400 nm for 3 hours, add 15 mL of cofactor NAD + After half an hour, 2 mL of the reaction solution was mixed with 0.3 mL of 2,4-dinitrophenylhydrazine, the pH was adjusted to 2-4, and the reaction was carried out in the dark for half an hour. The product content was then detected by HPLC to reflect the activity of alcohol dehydrogenase. At the same time, 2 mL of the reaction solution was taken into a spectrophotometer to detect the absorbance of NADH. The test results are shown in Table 4. The 10 fullerene derivatives in Table 4 can protect the activity of alcohol dehydrogenase to varying degrees under adverse light conditions. Among them, C 70 -OH and Sc3N@C80(C(COOH)2)2 have the best protection effect.

[0080] Table 4. Test of enzyme protection performance of different fullerene derivatives under 3 hours of light irradiation

[0081] Fullerene (derivative) <![CDATA[Absorbance A 340 > <![CDATA[C 70 -OH]]> 0.85 <![CDATA[C 60 -OH]]> 0.71 Hydroxyfullerene (THPC) 0.62 Aminofullerene (TAPC) 0.55 TCPC 0.52 <![CDATA[C 60 (C(COOH)2)2]]> 0.61 <![CDATA[C 70 (C(COOH)2)2]]> 0.67 <![CDATA[Sc3N@C 80 (C(COOH)2)2]]> 0.88 <![CDATA[PEG-C 60 ]]> 0.49 <![CDATA[Cyclodextrin-C 70 > 0.51

[0082] The effects of different fullerene derivatives in protecting and improving enzyme activity may be related to their conjugated parts, e.g., C 60 With C 70 Compared with the fullerene, it is a symmetrical structure, and the -OH groups connected are denser, which destroys the π conjugated structure of the fullerene. 70 When it is installed, only the equatorial plane is modified, resulting in C 70 The conjugation plane of -OH is larger, which means that the degree of conjugation is less destroyed by -OH, and the final result is C 70 -OH to C 60 -OH has a better effect on protecting enzymes. When metal fullerenes with larger carbon cages are selected, the degree of conjugation is less damaged under the same modification, that is, the more fully the conjugated structure is retained, the better the performance of the final protection enzyme.

[0083] It should be noted that not all water-soluble fullerene materials that can quench free radicals can protect enzymes. The present disclosure selects three water-soluble fullerene nanomaterials to test the protection of alcohol dehydrogenase under light damage conditions. The specific materials are sialic acid-modified functionalized C 70 Fullerene nanomaterials (STMF), alanine-modified fullerene nanomaterials (AFRM) and mannose-modified dicarboxyl fullerene derivatives (DCFM), the specific steps are the same as above, and the absorbance after the reaction is A 340 The summary is shown in Table 5.

[0084] Table 5. Fullerene (derivative) protection enzyme performance test under 3 hours of light

[0085] Fullerene (derivative) <![CDATA[Absorbance A 340 > <![CDATA[C 70 -OH]]> 0.85 STMF 0.17 AFRM 0.23 DCFM 0.13

[0086] These three materials have been proven to be able to scavenge free radicals, but they are not effective in protecting enzymes. The main reasons for this are that the R exogenous group structures of these three materials are very large. Taking the mannose-modified dicarboxyfullerene derivative (DCFM) as an example, the structure is described in the following formula (II). The overly large exogenous group at one end of the fullerene may change the spatial stereo configuration of the enzyme during the enzyme-catalyzed reaction, reduce the enzyme activity, or even destroy the structure of the enzyme. Not only does it fail to protect the enzyme, it even becomes a factor that destroys the enzyme activity, ultimately resulting in the inability to carry out the enzyme-catalyzed reaction.

[0087]

[0088] According to the above discussion on the mechanism of fullerene derivatives protecting enzymes, the protective and / or enhancing effect of fullerene derivatives on enzyme activity depends on the structure of the fullerene derivatives themselves, and is therefore not limited to specific enzyme types. For the convenience of detection, the present disclosure mainly selects dehydrogenase to verify the protective and / or enhancing effect of fullerene derivatives on enzyme activity.

[0089] Example 3. Protection of alcohol dehydrogenase by C70-OH in hydrogen peroxide with different concentrations

[0090] Control group: 0.3 mL of cofactor NAD was added to 0.9 mL of PBS solution containing 10 U of alcohol dehydrogenase. + and 50 μL of hydrogen peroxide solutions with concentrations of 2 mM, 1 mM, 500 μM, 250 μM and 0, respectively, were incubated for 0.5 h, and then 0.9 mL of 50% ethanol aqueous solution was added. The absorbance values ​​at 340 nm within 1 min were detected as A0 and A1, and the enzyme activity was calculated.

[0091] Experimental group: 0.1 mL of 0.8 mg / mL C 70 -OH, 0.3mL cofactor NAD+ and 50 μL of hydrogen peroxide solutions with concentrations of 2 mM, 1 mM, 500 μM, 250 μM and 0, respectively, were incubated for 0.5 h, and then 0.9 mL of ethanol was added. The absorbance values ​​at 340 nm within 1 min were detected as A0 and A1, and the enzyme activity was calculated.

[0092] Enzyme activity = A1-A0 / 1min

[0093] Each treatment group was set up with 3 replicates, and the results are shown in Figure 6 As shown. Figure 6 It can be seen that when the concentration of hydrogen peroxide is 2mM-250μM, C 70 -OH has a certain protective effect on alcohol dehydrogenase. The protective effect is most significant under 500 and 250μM hydrogen peroxide destruction. It is speculated that the enzyme activity is too severely damaged or even inactivated when the hydrogen peroxide concentration is 2 and 1mM. Under appropriate destruction conditions, when the enzyme activity is inhibited, C 70 -OH can increase its activity and accelerate the reaction rate.

[0094] Example 4.C 70 -OH protects alcohol dehydrogenase under different pH conditions

[0095] Control group: 0.3 mL of cofactor NAD was added to 0.1 mL of PBS solution containing 5 U of alcohol dehydrogenase. + and 0.8 mL of PBS solution with a pH of 6, 6.5, 7, 8, or 9, and incubated for 0.5 h. Then, 0.9 mL of 50% ethanol aqueous solution was added, and the absorbance at 340 nm within 1 min was detected as A0 and A1, and the enzyme activity was calculated.

[0096] Experimental group: 0.1 mL of 0.8 mg / mL C 70 -OH, 0.3mL cofactor NAD + and 0.7 mL of PBS solution with a pH of 6, 6.5, 7, 8, or 9, and incubated for 0.5 h. Then, 0.9 mL of 50% ethanol aqueous solution was added, and the absorbance at 340 nm within 1 min was detected as A0 and A1, and the enzyme activity was calculated.

[0097] Enzyme activity = A1-A0 / 1min

[0098] Each treatment group was set up with 3 replicates. The optimal catalytic environment for alcohol dehydrogenase is pH=8.7. Figure 7 It is C 70 Schematic diagram of the protective ability of -OH on alcohol dehydrogenase under different pH environments. Under the optimal reaction conditions, C 70-OH can significantly increase its activity and accelerate the reaction rate. Under other destructive conditions, when the activity of the enzyme is inhibited but not inactivated, C 70 -OH can also play a certain protective role.

[0099] Example 5.C 60 -OH protects acetaldehyde dehydrogenase under different pH conditions

[0100] The acetaldehyde dehydrogenase activity was detected using Nanjing Jiancheng acetaldehyde dehydrogenase activity detection kit: acetaldehyde dehydrogenase catalyzes the reaction of acetaldehyde and NAD + When converted into acetic acid and NADH, the absorbance at 340nm will increase. When testing, take 0.1mL of treated enzyme solution and mix thoroughly with 0.9mL of working solution, read A1, and after incubation at 37℃ for 5min, read A2.

[0101] The kit catalyzes the reduction of 1 nmol NAD per minute per mL of sample. + The amount of enzyme is defined as 1 unit of enzyme activity.

[0102] Acetaldehyde dehydrogenase activity = ΔA*V 反总 / ε*d*V 样 *T=322*ΔA

[0103] Among them, V 反总 : total volume of the reaction system, 1 mL; ε: molar extinction coefficient of NADH, 6.22*10 -3 L / μmol / cm; d: cuvette light path, 1 cm; V 样 : volume of extract added, 1 mL; T: reaction time, 5 min.

[0104] The control group was prepared by adding 0.5 mL of PBS solution with a pH of 6, 6.5, 7, 8 or 9 and 0.5 mL of deionized water to 1 mL of PBS solution containing 20 U of acetaldehyde dehydrogenase. After incubation for 0.5 h, 0.1 mL was taken out and mixed with 0.9 mL of working solution. A1 was read. After incubation at 37°C for 5 min, A2 was read and the enzyme activity was calculated.

[0105] The experimental group was composed of 1 mL of PBS solution containing 20 U of acetaldehyde dehydrogenase, 0.5 mL of PBS solution with a pH of 6, 6.5, 7, 8, or 9 and 0.5 mL of C at a concentration of 0.8 mg / ml. 60 -OH, after incubation for 0.5h, take 0.1mL and 0.9mL working solution and mix thoroughly, read A1, incubate at 37℃ for 5min, read A2 and calculate the enzyme activity.

[0106] Each treatment group was set up with 3 replicates. Figure 8 It is C 60-OH protection performance test results of acetaldehyde dehydrogenase under different pH environments show that the best catalytic environment for acetaldehyde dehydrogenase is pH = 7.9. Near the best reaction conditions (7-8), C 60 -OH can significantly improve its activity and accelerate the reaction rate.

[0107] Example 6.C 60 -OH protects acetaldehyde dehydrogenase in hydrogen peroxide with different concentrations

[0108] The Nanjing Jiancheng acetaldehyde dehydrogenase activity kit was used to detect the activity of acetaldehyde dehydrogenase: acetaldehyde dehydrogenase catalyzes the reaction of acetaldehyde and NAD + When converted into acetic acid and NADH, the absorbance at 340nm will increase. When testing, take 0.1mL of treated enzyme solution and mix thoroughly with 0.9mL of working solution, read A1, and after incubation at 37℃ for 5min, read A2.

[0109] The control group was prepared by adding 50 μL of 0.25, 0.5, 1 or 2 mM hydrogen peroxide solution and 0.5 mL of deionized water to 1 mL of PBS solution containing 20 U of acetaldehyde dehydrogenase. After incubation for 0.5 h, 0.1 mL was taken out and mixed with 0.9 mL of working solution. A1 was read. After incubation at 37°C for 5 min, A2 was read and the enzyme activity was calculated.

[0110] The experimental group was composed of 1 mL of PBS solution containing 20 U of aldehyde dehydrogenase, 50 μL of 0.25, 0.5, 1 or 2 mM hydrogen peroxide solution and 0.5 mL of 0.8 mg / ml C 60 -OH, after incubation for 0.5h, take 0.1mL and 0.9mL working solution and mix thoroughly, read A1, incubate at 37℃ for 5min, read A2 and calculate the enzyme activity.

[0111] Acetaldehyde dehydrogenase activity = ΔA*V 反总 / ε*d*V 样 *T=322*ΔA

[0112] Each treatment group was set up with 3 replicates, and the test results showed that: 60 -OH had a significant protective effect on acetaldehyde dehydrogenase in the range of 500-250 μM, with an enzyme activity of 3.9, close to the blank group (enzyme activity of 4.5).

[0113] Example 7. Protection of acetaldehyde dehydrogenase by aminofullerene (TAPC) under different pH conditions

[0114] Aminofullerene (TAPC) was obtained from the laboratory and has the following structural formula:

[0115]

[0116] TAPC was prepared according to the method disclosed in the prior art (e.g., CN112516164A, the content of which is incorporated herein by reference), and the specific preparation method is as follows:

[0117] 5g C 60 Dissolve in 625mL chlorobenzene, add 13g 4-(tert-butyloxycarbonylamino)piperidine and 5mL isopropylbenzene hydroperoxide (80%), stir at room temperature (600rpm) for 48h, dilute the reaction solution 10 times and perform HPLC detection. When the reaction is complete, stop the reaction, wash the reaction solution with 300mL saturated ammonium chloride solution and 300mL saturated sodium bicarbonate solution in turn, dry the organic layer with anhydrous sodium sulfate, concentrate and evaporate to dryness at 60℃ to obtain a reddish brown solid. Dissolve the reaction product in toluene and perform column chromatography separation, the eluent is ethyl acetate / toluene 10%, 12%, 15%, 20%. Separate and obtain tert-butyloxycarbonyl protected tri-addition and tetra-addition aminofullerene samples. Add 375mL TFA / CHCl3 (10%) to 1g of the separated aminofullerene sample, stir at room temperature for 2h, concentrate and evaporate to dryness to obtain a red solid (98.5%), dissolve the solid in water and perform dialyzation, and freeze-dry to obtain a deprotected red solid. The deprotected sample was dissolved in water and passed through an ion exchange column for three times. The sample remaining in the column was eluted with water. The sample aqueous solution was filtered through a 0.22 μm filter membrane, dialyzed, and freeze-dried to obtain the TAPC product used in the present disclosure.

[0118] The control group was prepared by adding 0.5 mL of PBS solution with a pH of 6, 6.5, 7, 8 or 9 and 0.5 mL of deionized water to 1 mL of PBS solution containing 20 U of acetaldehyde dehydrogenase. After incubation for 0.5 h, 0.1 mL was taken out and mixed with 0.9 mL of working solution. A1 was read. After incubation at 37°C for 5 min, A2 was read and the enzyme activity was calculated.

[0119] In the experimental group, 0.5 mL of PBS solution with a pH of 6, 6.5, 7, 8 or 9 and 0.5 mL of TAPC with a concentration of 0.8 mg / mL were added to 1 mL of PBS solution containing 20 U of acetaldehyde dehydrogenase. After incubation for 0.5 h, 0.1 mL was taken out and mixed with 0.9 mL of working solution. A1 was read. After incubation at 37°C for 5 min, A2 was read and the enzyme activity was calculated.

[0120] Acetaldehyde dehydrogenase activity = ΔA*V 反总 / ε*d*V 样 *T=322*ΔA

[0121] Three replicates were set for each treatment group. The test results showed that fullerene (TAPC) had a significant protective effect on acetaldehyde dehydrogenase in the pH range of 7-8, with an enzyme activity of 4.1, close to the blank group (enzyme activity of 4.5).

[0122] Example 8. Protection of acetaldehyde dehydrogenase by carboxyl fullerene (TCPC) in hydrogen peroxide with different concentrations

[0123] TCPC was obtained from the laboratory and has the following structural formula:

[0124]

[0125] TCPC was prepared according to the method disclosed in the prior art (e.g., Huo, J. et al., Amphiphilic Aminated Derivatives of

[60] Fullerene as Potent Inhibitors of Tumor Growth and Metastasis.

[0126] Advanced Science 2022, 2201541, the contents of which are incorporated herein by reference), the specific preparation method is as follows:

[0127] C 60 (5 g) and 4-N-BOC-aminopiperidine (13 g) were dissolved in chlorobenzene (625 mL), and cumene hydroperoxide (5 mL, 80%) was added. The reaction progress was monitored by HPLC. After stirring at room temperature (600 rpm) for 48 h, C 60 It was basically completely consumed. The reaction was stopped with dimethyl sulfide (10 mL), washed with a saturated aqueous solution of NH4Cl (300 mL) and NaHHCO3 (300 mL), and dried over anhydrous Na2SO4. The crude product was evaporated at 60°C as a reddish-brown solid. Boc-protected TCPC was purified by silica gel column chromatography (Rf: 0.3, toluene solution containing 25-30% ethyl acetate as the mobile phase). The tert-butyl group was removed by stirring in 50% TFA / CHCl3 at room temperature for 6 hours, and then dissolved in an aqueous solution of NaOH (100 μM) after drying, and dialyzed with ultrapure water for 48 hours, and vacuum dried to obtain a water-soluble fullerene derivative TCPC.

[0128] The control group was prepared by adding 50 μL of 0.25, 0.5, 1 or 2 mM hydrogen peroxide solution and 0.5 mL of deionized water to 1 mL of PBS solution containing 20 U of acetaldehyde dehydrogenase. After incubation for 0.5 h, 0.1 mL was taken out and mixed with 0.9 mL of working solution. A1 was read. After incubation at 37°C for 5 min, A2 was read and the enzyme activity was calculated.

[0129] In the experimental group, 50 μL of 0.25, 0.5, 1 or 2 mM hydrogen peroxide solution and 0.5 mL of 0.8 mg / mL TCPC were added to 1 mL of PBS solution containing 20 U of acetaldehyde dehydrogenase. After incubation for 0.5 h, 0.1 mL was taken out and mixed with 0.9 mL of working solution. A1 was read. After incubation at 37°C for 5 min, A2 was read and the enzyme activity was calculated.

[0130] Acetaldehyde dehydrogenase activity = ΔA*V 反总 / ε*d*V*T=322*ΔA

[0131] Three replicates were set for each treatment group. The test results showed that TCPC had a significant protective effect on acetaldehyde dehydrogenase in the range of 500-250 μM, with an enzyme activity of 4.3, close to the blank group (enzyme activity of 4.5).

[0132] Example 9. Hydroxyfullerene (THPC) protects alcohol dehydrogenase in hydrogen peroxide with different concentrations

[0133] According to the preparation methods of Examples 7 and 8, fullerene derivatives with surface hydroxyl groups were synthesized to explore the protective effect under the damage of hydrogen peroxide at different concentrations. The structural formula of hydroxyfullerene (THPC) is as follows:

[0134]

[0135] Control group: 0.3 mL of cofactor NAD was added to 0.9 mL of PBS solution containing 10 U of alcohol dehydrogenase. + and 50 μL of 0.25, 0.5, 1 or 2 mM hydrogen peroxide solution, incubated for 0.5 h, then added 0.9 mL of ethanol and reacted for 30 min. 340 .

[0136] Experimental group: 0.1 mL of 0.8 mg / mL THPC and 0.3 mL of cofactor NAD were added to 0.9 mL of PBS solution containing 10 U of alcohol dehydrogenase. + and 50 μL of 0.25, 0.5, 1 or 2 mM hydrogen peroxide solution, incubated for 0.5 h, then added 0.9 mL of 50% ethanol solution, reacted for 30 min, and measured A 340 .

[0137] The two were compared to determine whether THPC has a protective effect. Three replicates were set for each treatment group. The test results showed that: under appropriate hydrogen peroxide destruction (500-250μM), the experimental group reacted for 30 minutes and then A 340 It is about 0.8, close to the value A when the reaction is complete 340 =0.88.

[0138] Example 10. Polyethylene glycol-modified fullerene nanomaterials (PEG-C60) protect alcohol dehydrogenase in hydrogen peroxide with different concentrations

[0139] Control group: 0.3 mL of cofactor NAD was added to 0.9 mL of PBS solution containing 10 U of alcohol dehydrogenase. + and 50 μL of 0.25, 0.5, 1 or 2 mM hydrogen peroxide solution, incubated for 0.5 h, then added 0.9 mL of ethanol and reacted for 30 min. 340 .

[0140] Experimental group: 0.1 mL of 0.8 mg / mL PEG-C was added to 0.9 mL of PBS solution containing 10 U of alcohol dehydrogenase. 60 、0.3mL cofactor NAD + and 50 μL of 0.25, 0.5, 1 or 2 mM hydrogen peroxide solution, incubated for 0.5 h, then added 0.9 mL of ethanol and reacted for 30 min. 340 .

[0141] Compare the two to determine whether PEG-C 60 Each treatment group was set up with 3 replicates, and the test results showed that: under the destruction of 500-250μσM hydrogen peroxide, PEG-C 60 A of group reaction 30min 340 =0.58, close to the value A when the enzyme catalyzes the reaction completely 340 =0.88, while the control group's A 340 =0.15, almost no reaction occurred, which indicates that under the destruction of appropriate concentration of hydrogen peroxide, PEG-C 60 It has a certain protective effect on alcohol dehydrogenase.

[0142] Example 11. Protection of alcohol dehydrogenase by different fullerene derivatives in hydrogen peroxide with different concentrations

[0143] We also used different fullerenes (derivatives) to compare the protection ability of the same enzyme. Here we chose alcohol dehydrogenase. The specific steps are as shown in Example 10. The specific data are shown in Table 6. It can be seen that after 30 minutes of reaction, the fullerene derivative material has a significant protective effect on the enzyme.

[0144] Table 6. Protection of alcohol dehydrogenase by different fullerene derivatives under hydrogen peroxide destruction

[0145] Fullerene (derivative) Enzymes Substrate <![CDATA[Absorbance A 340 > <![CDATA[C 70 -OH]]> Alcohol dehydrogenase Ethanol 0.85 <![CDATA[Sc3N@C 80 (C(COOH)2)2]]> Alcohol dehydrogenase Ethanol 0.88 TCPC Alcohol dehydrogenase Ethanol 0.65 <![CDATA[Cyclodextrin-C 60 > Alcohol dehydrogenase Ethanol 0.51 <![CDATA[Hyaluronic acid-C 70 > Alcohol dehydrogenase Ethanol 0.54

[0146] Example 12. Protection of different enzymes by different fullerene derivatives in hydrogen peroxide of different concentrations

[0147] At the same time, we also used different enzymes and corresponding substrates to verify the universality of different fullerene derivative materials to protect enzymes. The specific steps refer to Example 10, and the specific data are shown in Table 7. It can be seen that after 30 minutes of reaction, the A of the fullerene derivative material group 340 Both are close to the values ​​when the enzyme catalyzed reaction is complete.

[0148] Table 7. Universality of protective enzyme verified by different materials and different enzymes

[0149]

[0150] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present disclosure, rather than to limit the protection scope of the present disclosure. Simple modifications or equivalent substitutions of the technical solution of the present disclosure by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present disclosure.

Claims

1. Use of the compound represented by formula (I) in protecting and / or improving enzyme activity, in, In formula (I), F is a fullerene, and the fullerene is selected from at least one of hollow fullerene, metallofullerene, heterocyclic fullerene and endohedral fullerene; R is a modification group of the fullerene, and the modification group is a modification group used to increase water solubility; m is the number of modifying groups, 1≤m≤30.

2. An enzyme protecting agent, comprising a compound represented by formula (I).

3. A medicine, food, health product and / or cosmetic comprising the compound represented by formula (I), which also comprises an enzyme.

4. The use according to claim 1, the enzyme protectant according to claim 2, or the medicine, food, health product and / or cosmetic according to claim 3, wherein: The compounds protect and / or enhance enzyme activity under adverse conditions; Preferably, the unfavorable conditions include temperature, light, pH value and / or free radical concentration that are unfavorable for enzyme activity; More preferably, the unfavorable conditions include light, pH value and / or free radical concentration that are unfavorable for enzyme activity; Further preferably, the unfavorable conditions include a light exposure time of 1-12 hours, a pH value of 3-10 and / or a free radical concentration range of 0.25-2 mM.

5. The use according to claim 1 or 4, the enzyme protecting agent according to claim 2 or 4, or the medicine, food, health product and / or cosmetic according to claim 3 or 4, wherein: One end of the R is bonded to the fullerene via an alkyl chain, a nitrogen-containing group, a phenyl group or a mercapto group, and the other end of R is a group containing a carboxyl group, an amino group and / or a hydroxyl group; Preferably, when the other end of R is a hydroxyl group, 1≤m≤4 or 15≤m≤30; Preferably, when the other end of R is a group other than a hydroxyl group, 1≤m≤4.

6. The use according to any one of claims 1, 4-5, the enzyme protectant according to any one of claims 2, 4-5, or the medicine, food, health product and / or cosmetic according to any one of claims 3-5, wherein: The fullerene is selected from hollow fullerene C 2a 、Metallofullerene A2C2@C 2b or at least one of metallofullerene B3N@C2b, wherein 30≤a≤50, 39≤b≤44, A is at least one of Sc, La, and Y, and B is at least one of Sc, La, Y, Ho, Lu, and Er; Preferably, a is 30, 35, 38, 39, 41 or 42; Preferably, b is 41 or 42.

7. The use according to any one of claims 1 and 4 to 6, the enzyme protectant according to any one of claims 2 and 4 to 6, or the medicine, food, health product and / or cosmetic according to any one of claims 3 to 6, wherein: The compound is selected from C 70 -OH, C 60 -OH, aminofullerene (TAPC), carboxyfullerene (TCPC), hydroxyfullerene (THPC), C 60 (C(COOH)2)2、C 70 (C(COOH)2)2, Sc3N@C80(C(COOH)2)2, hyaluronic acid modified fullerene nanomaterials (HA-C 60 ), cyclodextrin-modified fullerene nanomaterials, polyethylene glycol-modified fullerene nanomaterials (PEG-C 60 ), polyvinylpyrrolidone modified fullerene nanomaterials (PVP-C 60 ), polyvinylpyrrolidone-modified fullerene nanomaterials (PVA-C 60 ) at least one of.

8. The use according to any one of claims 1 and 4 to 7, the enzyme protectant according to any one of claims 2 and 4 to 7, or the medicine, food, health product and / or cosmetic according to any one of claims 3 to 7, wherein: The concentration of the compound is 0.2-2 mg / mL; Preferably, the concentration of the compound is 0.8-1.5 mg / mL; More preferably, the concentration of the compound is 0.8 mg / mL.

9. The use according to any one of claims 1 and 4 to 8, the enzyme protectant according to any one of claims 2 and 4 to 8, or the medicine, food, health product and / or cosmetic according to any one of claims 3 to 8, wherein: The enzyme is selected from at least one of glucose-6-phosphate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, acetaldehyde dehydrogenase, α-ketoglutarate dehydrogenase, enolone dehydrogenase and formate dehydrogenase; Preferably, the enzyme is alcohol dehydrogenase, acetaldehyde dehydrogenase and / or formate dehydrogenase; Preferably, when the enzyme is alcohol dehydrogenase, the general formula of its substrate is S-OH, wherein S is selected from -CH3, -C2H5, -C4H9, -C5H 11 , -CH(CH3)2, -CH2CH(CH3)2, -CH2C6H5 and -CH2CH2OH; More preferably, S is at least one selected from -CH3, -C2H5 and -CH2C6H5; Preferably, when the enzyme is acetaldehyde dehydrogenase, its substrate is CH3CHO and / or C2H5CHO; more preferably, the substrate is CH3CHO; Preferably, when the enzyme is formate dehydrogenase, its substrate is HCOOH.

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