Application of polymer coupled with flavin group in catalysis of conversion of NADH (nicotinamide adenine dinucleotide) in reduction state into NAD + (nicotinamide adenine dinucleotide) in oxidation state

Abstract

The invention discloses application of a polymer coupled with a flavin group to catalysis of conversion of NADH (nicotinamide adenine dinucleotide) in a reduction state into NAD + (nicotinamide adenine dinucleotide) in an oxidation state, which is characterized in that in a solution containing various components, the polymer coupled with the flavin group catalyzes the NADH to convert the NAD +, the conversion rate is 0.007-0.85 mu M / s, and the conversion efficiency is 20-100%; the polymer coupled with the flavin group is a product obtained by coupling flavin with a polymer containing a cationic group side group through a covalent bond, the molar ratio of the flavin group in a product molecule to the cationic group in the polymer is 1: 1-1: 100, and the polymer is composed of a repetitive unit containing the cationic group and other repetitive units. Wherein the molar fraction of the amino-containing repeating unit is 5%-100%. Compared with riboflavin, flavin mononucleotide and other biomolecules containing flavin groups, the activity of catalyzing NADH to be oxidized into NAD + can be improved by at least 50 times or above.

Description

Technical Field

[0001] The present invention belongs to the field of bionic catalysis and relates to a polymer coupled with a flavin group in catalyzing the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD + Applications on. Background Art

[0002] Oxidized nicotinamide adenine dinucleotide (NAD + ) plays an important role in cell repair, anti-aging, skin regeneration and anti-oxidation. As people's demand for health and beauty increases, NAD + Related concepts have gradually been introduced into skin care products to enhance skin cell vitality, reduce fine lines and wrinkles, and improve skin tone. With the layout of major beauty companies, NAD, which is known for "activating cell energy and delaying skin aging", has become a hot topic in the cosmetics industry. + Standing on the new outlet of the beauty industry. In early 2024, L'Oréal launched the Lancome Black Gold series, which also focuses on NAD + Launch product promotion, emphasizing "reverse aging, youth". However, the existing oral (latest regulations: NMN / NAD from some countries + Oral products have been banned), injections, and methods that stimulate cell regeneration also have problems with low bioavailability, low transdermal efficiency, and side effects.

[0003] NAD + The balance of NAD / NADH is considered an important parameter for the health of cells. + The level of NAD + The ratio of NAD to NADH decreases, which is usually accompanied by the accumulation of NADH due to insufficient NAD+ regeneration. In addition, studies have shown that with aging, the NADH dehydrogenase (which catalyzes the conversion of NADH to NAD+) in cells + ) expression levels decreased. However, there are no mainstream cosmetic brands that supplement with catalysts that have NADH dehydrogenase function. Summary of the invention

[0004] In view of this, the present invention provides a polymer coupled with a flavin group for catalyzing the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD + The polymer coupled with flavin groups mimics the function of NADH dehydrogenase, catalyzing the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD + The present invention specifically provides the following technical solutions:

[0005] The invention discloses an application of a polymer coupled with a flavin group in catalyzing the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD+. In a solution containing multiple components, the polymer coupled with a flavin group catalyzes the conversion of NADH into NAD+, with a conversion rate of 0.007 μM / s to 0.85 μM / s and a conversion efficiency of 20% to 100%. The polymer coupled with a flavin group is a product of flavin coupled with a polymer containing a cationic group side group through a covalent bond, the molar ratio of the flavin group in the product molecule to the cationic group in the polymer is 1:1 to 1:100, and the polymer is composed of repeating units containing cationic groups and other repeating units, wherein the molar fraction of the repeating units containing cationic groups is 5% to 100%.

[0006] Further, the polymer coupled with flavin groups catalyzes the conversion of NADH to NAD + At the same time, it can also reduce the oxidized coenzymes Q0 and Q10.

[0007] Furthermore, the cationic group is a protonated amine, a guanidine group, a quaternary ammonium salt, a protonated pyridine, a protonated imidazole, or a triphenylphosphine cation.

[0008] Furthermore, the cationic group-containing polymer is a polypeptide, polyallylamine or chitosan.

[0009] Furthermore, the polymer coupled with the flavin group is a flavin / polyallylamine conjugate, and the structural formula is Wherein, n = 800-900;

[0010] or flavin / poly((glutamine) 10 -(α-Lysine) 10 ) conjugate, the structural formula is:

[0011] Or flavin / chitosan conjugate, the structural formula is: Wherein, n = 730 ~ 1500;

[0012] or flavin / poly((α-lysine)5-(arginine) 15 ) conjugate, the structural formula is:

[0013] Furthermore, the solution of multiple components is a phosphate buffer solution.

[0014] Furthermore, the solution of multiple components is uric acid or glutathione or ascorbic acid or glutathione / uric acid mixture solution.

[0015] Furthermore, the solution of multiple components is a NaCl solution.

[0016] Furthermore, the solution of multiple components is a culture medium.

[0017] Furthermore, the culture medium composition is 90% MEM+10% FBS+1% PS.

[0018] Furthermore, the FBS is derived from fetal bovine serum, and PS contains penicillin-streptomycin dual antibodies.

[0019] The beneficial effects of the present invention are as follows: the present invention uses flavin molecules (vitamin B2 derivatives) and biocompatible macromolecules as raw materials to synthesize NADH dehydrogenase-like catalyst materials, which can not only effectively and specifically catalyze NADH oxidation in aqueous solution and cell culture medium environment to achieve NAD + The present invention modifies the flavin group on the surface of a polymer with a cationic charge (amino group) through a covalent bond, and promotes NADH to approach the flavin catalytic center through the electrostatic attraction between the cationic charge on the amino group and the negative charge of the phosphate on the nicotinamide adenine dinucleotide (NADH), simulating the activity of NADH dehydrogenase, and catalyzing the oxidation of nicotinamide adenine dinucleotide (NADH) to an oxidized state (NAD + ). This covalent coupling constructs enzyme-like catalysts. Compared with the physical blend of flavin molecules / polymers, the flavin groups are firmly attached to the polymer surface to form a stable catalytic system, so that the flavin groups can continue to play a catalytic role during the reaction and will not become ineffective due to detachment. Compared with biological molecules containing flavin groups such as riboflavin and flavin mononucleotides, it catalyzes the oxidation of NADH to NAD + The activity can be increased by at least 50 times; compared with the blend of flavin molecules / polymers, the catalytic activity is increased by nearly an order of magnitude. In addition, when the flavin-modified polymer catalyst is used in cosmetic raw materials, this stability ensures that the flavin catalytic group is not interfered by other substances in the biological system after it penetrates into the skin layer, thereby destroying the interaction between the flavin group and the polymer, ensuring that the catalyst can continue to work effectively and is more conducive to improving cell vitality. The catalyst of the present invention can be used as a substitute for natural NADH dehydrogenase, and in principle can avoid other NAD + Reduce the side effects of supplementation and achieve anti-aging effects on the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings:

[0021] Figure 1 Flavin / poly((glutamine) 10 -(α-Lysine)10 ) UV-visible absorption spectra of the conjugate and flavin mononucleotide.

[0022] Figure 2 Flavin / poly((glutamine) 10 -(α-Lysine) 10 ) The conjugates and blends catalyze the conversion of NADH to NAD + The rate diagram.

[0023] Figure 3 Flavin / poly((glutamine) 10 -(α-Lysine) 10 ) The conjugate catalyzes the production of NAD + Mass spectrum of .

[0024] Figure 4 For various reducing agent molecular components to flavin / poly((glutamine) 10 -(α-Lysine) 10 )Effect diagram of the conjugate activity.

[0025] Figure 5 NaCl to flavin / poly((glutamine) 10 -(α-Lysine) 10 )Effect diagram of the conjugate activity.

[0026] Figure 6 Flavin / poly((glutamine) 10 -(α-Lysine) 10 ) Data of catalytic reduction of coenzyme Q0 by the conjugate.

[0027] Figure 7 Flavin / poly((glutamine) 10 -(α-Lysine) 10 ) Catalytic data of the conjugate in culture medium.

[0028] Figure 8 The UV-visible absorption spectra of flavin / polyallylamine conjugate and flavin mononucleotide.

[0029] Fig. 9 This is a comparison chart of the activities of flavin / polyallylamine conjugates and blends.

[0030] Fig.10 Flavin mononucleotide / poly((α-lysine)5-(arginine) 15 ) UV-visible absorption spectra of the conjugate and flavin mononucleotide

[0031] Fig.11 Flavin mononucleotide / poly((α-lysine)5-(arginine) 15)Comparison of the activities of the conjugate and the blend.

[0032] Fig.12 The UV-visible absorption spectra of flavin / chitosan conjugate and flavin mononucleotide.

[0033] Fig.13 This is a comparison chart of the activities of flavin / chitosan conjugates and blends. DETAILED DESCRIPTION

[0034] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings.

[0035] Example 1

[0036] 1. Preparation of flavin / poly((glutamine) 10 -(α-Lysine) 10 )Conjugate

[0037] Riboflavin was oxidized to aldehyde-flavin molecules by periodic acid oxidation reaction, and the product was separated and purified to obtain yellow powder with a yield of about 50%. Subsequently, the aldehyde-flavin molecules were reacted with poly((glutamine) 10 -(α-Lysine) 10 ) (In this polypeptide, there are 10 glutamines and lysines respectively, and the molar fraction of the amino repeating unit is 50%). The riboflavin / poly((glutamine)) was coupled by condensation and reductive amination reaction, and purified by dialysis to obtain riboflavin / poly((glutamine)) 10 -(α-Lysine) 10 ) conjugate, freeze-dried, powder sample stored at 2-8℃ away from light. Riboflavin / poly((glutamine) 10 -(α-Lysine) 10 ) The structural formula of the conjugate is:

[0038]

[0039] 2. Flavin / Poly((Glutamine) 10 -(α-Lysine) 10 ) Chemical structure characterization of conjugates

[0040] UV-Vis spectroscopy was used to characterize the flavin / poly((glutamine) 10 -(α-Lysine) 10 ) structural characterization. Figure 2 Flavin / poly((glutamine) 10 -(α-Lysine) 10 ) conjugate and flavin mononucleotide. Figure 2 It can be determined that the flavin group is covalently coupled to ε-polylysine, wherein the molar ratio of flavin to lysine is 1 / 10.

[0041] 3. Flavin mononucleotide / poly((glutamine) 10 -(α-Lysine) 10 ) Preparation of blends

[0042] Considering the interaction between flavin mononucleotide and poly((glutamine) 10 -(α-Lysine) 10 ) possible electrostatic interaction, and they were mixed as a control group. 10 -(α-Lysine) 10 ) are mixed in water to ensure that the molar ratio of flavin to α-lysine is 1 / 10, and then freeze-dried, and the freeze-dried powder sample is stored at 2-8°C in the dark.

[0043] 4. Flavin / Poly((Glutamine) 10 -(α-Lysine) 10 ) The conjugate acts as a catalyst to catalyze the conversion of NADH to NAD + Speed ​​test

[0044] (1) Dissolve flavin / poly((glutamine)) in ultrapure water 10 -(α-Lysine) 10 ) conjugate to obtain a solution with a flavin concentration of 100 μM, which was stored in the dark at room temperature after preparation;

[0045] (2) Dissolve NADH in ultrapure water to obtain a 10 mM solution. After preparation, store the solution at 2-8°C.

[0046] Take 1 μl of solution (1) and (2) respectively and add them into a micro-cuvette containing 98 μl of 50 mM phosphate buffer, mix them quickly and evenly, measure the change of absorbance at 340 nm, and record the flavin / poly((glutamine)) 10 -(α-Lysine) 10 ) as a catalyst to catalyze the conversion of NADH to NAD + The rate of flavin mononucleotide / poly((glutamine) 10 -(α-Lysine) 10 ) blend was used as the control group.

[0047] Figure 2 Catalyzes the conversion of NADH to NAD + Rate diagram of poly((glutamine) 10 -(α-Lysine) 10 ) is referred to as poly((Q) 10 -(α-K) 10 ).from Figure 2 It can be seen that flavin / poly((glutamine)10 -(α-Lysine) 10 ) conjugate catalyst and flavin mononucleotide / poly((glutamine) 10 -(α-Lysine) 10 ) blend and flavin mononucleotide, the catalytic activity was 0.22 μM s -1 ,0.0192μM s -1 , and 0.0014 μM s -1 , indicating that flavin and poly((glutamine) 10 -(α-Lysine) 10 )After covalent coupling, the catalytic activity of the flavin group was greatly enhanced.

[0048] 5. Product testing of catalytic NADH oxidation

[0049] Determination of NAD using mass spectrometry + The formation of products. Figure 3 To generate NAD + The mass spectrum of Figure 3 As can be seen, the mass-to-charge ratio (664.126) is catalyzed by the oxidation of NADH to NAD + This indicates that the catalyst of the present invention can promote the conversion of NADH into NAD + .

[0050] 6. Catalytic activity test in the presence of ascorbic acid (AA), uric acid (UA), glutathione (GSH) or a mixture of ascorbic acid / uric acid / glutathione

[0051] Take 1 μl of the solution of step 4 (1) and (2) respectively and add them into a micro-cuvette containing 98 μl of 50 mM phosphate buffer (containing 1 mM uric acid, 1 mM glutathione, 1 mM ascorbic acid or 1 mM glutathione / 1 mM uric acid mixture), mix them quickly and evenly, measure the change of absorbance at 340 nm, and record the concentration of flavin / poly((glutamine)) 10 -(α-Lysine) 10 ) Catalyst catalyzes the conversion of NADH to NAD + rate.

[0052] Figure 4 For various reducing agent molecular components to flavin / poly((glutamine) 10 -(α-Lysine) 10 ) The effect of the catalytic activity of the conjugate, Figure 4 In the present invention, the conjugate catalyst represents flavin / poly((glutamine) 10 -(α-Lysine) 10) conjugate, AA represents ascorbic acid, UA represents uric acid, GSH represents glutathione, and GSH / UA / AA represents a mixture of glutathione / uric acid / ascorbic acid. Figure 4 As can be seen from the figure, the above ingredients (ascorbic acid, uric acid, glutathione or ascorbic acid / uric acid / glutathione mixture), flavin / poly((glutamine) 10 -(α-Lysine) 10 ) The activity of the conjugate catalyst is still largely retained, and even when glutathione is added, the activity is improved to a certain extent.

[0053] 7. Catalytic rate test in the presence of different concentrations of NaCl

[0054] Take 1 μl of the solution of step 4 (1) and (2) respectively and add them into a micro-cuvette containing 98 μl of 50 mM phosphate buffer (containing different concentrations of NaCl), mix them quickly and evenly, measure the change of absorbance at 340 nm, and record the riboflavin / poly((glutamine)) 10 -(α-Lysine) 10 ) The conjugate catalyst catalyzes the conversion of NADH to NAD + rate.

[0055] Figure 5 This is the effect of NaCl on the catalyst. Figure 5 It can be seen from the activity data that the addition of different concentrations of NaCl can reduce the activity of flavin / poly((glutamine) 10 -(α-Lysine) 10 ) The catalytic activity of the conjugate is reduced, but more than 35% can be retained.

[0056] 8. Test of catalytic reduction rate of coenzyme Q0

[0057] Dissolve oxidized coenzyme Q0 in ultrapure water to obtain a solution with a concentration of 1 mM. After preparation, store it at 2-8°C. Take 1 μl of the solution obtained from step 4 (1), (2) and coenzyme Q0 solution, add them to a micro-cuvette containing 97 μl of 50 mM phosphate buffer, mix quickly and evenly, and use high performance liquid chromatography to determine the concentration of flavin / poly((glutamine)) 10 -(α-Lysine) 10 )The reduction rate of coenzyme Q0 by the conjugate catalyst.

[0058] Figure 6 For single NADH, flavin / poly((glutamine) 10 -(α-Lysine) 10 ) The data diagram of converting the oxidized state of coenzyme Q0 to the reduced state when the conjugate and NADH coexist. Figure 6Poly((glutamine) 10 -(α-Lysine) 10 ) is referred to as poly((Q) 10 -(α-K) 10 ).from Figure 6 The activity data show that compared with the solution with only NADH, the flavin / poly((glutamine) 10 -(α-Lysine) 10 ) coupling catalyst and NADH co-exist, the oxidized coenzyme Q0 can be reduced at a faster rate, confirming that flavin / poly((glutamine) 10 -(α-Lysine) 10 )The conjugate catalyst catalyzes the reduction of coenzyme Q0.

[0059] 9. Test of the oxidation rate of NADH catalyzed under cell culture medium conditions

[0060] Take 1 μl of the solution of step 4 (1) and (2) respectively and put them into a micro-cuvette containing 98 μl of culture medium: 90% MEM + 10% FBS (serum from fetal bovine) + 1% PS (penicillin-streptomycin, double antibody), mix them quickly and evenly, measure the change of absorbance at 340 nm, and record the concentration of flavin / poly((glutamine)) 10 -(α-Lysine) 10 ) The conjugate catalyzes the conversion of NADH to NAD + rate.

[0061] Figure 7 Flavin / poly((glutamine) 10 -(α-Lysine) 10 ) The catalytic data of the conjugate in the culture medium, Figure 7 Poly((glutamine) 10 -(α-Lysine) 10 ) is referred to as poly((Q) 10 -(α-K) 10 ).from Figure 7 The activity data show that in cell culture medium containing a variety of complex components, flavin / poly((glutamine) 10 -(α-Lysine) 10 )The conjugate catalyst can still promote the oxidation reaction of NADH.

[0062] Example 2

[0063] 1. Preparation of flavin / polyallylamine conjugate

[0064] N,N'-Carbonyldiimidazole was used as a coupling activation reagent to couple riboflavin with polyallylamine (average M w50,000) in DMSO solvent, react at 25 degrees Celsius for 5 hours, and then dialysis purification to obtain riboflavin / polyallylamine, freeze-dried, and the powder sample is stored at 2-8 degrees Celsius in the dark. The structural formula of riboflavin / polyallylamine is:.

[0065] Where n is 800~900.

[0066] 2. Chemical structure characterization of flavin / polyallylamine coupling catalyst

[0067] Its structure was characterized by UV-visible spectroscopy. Figure 8 The UV-visible absorption spectra of flavin / polyallylamine conjugate and flavin mononucleotide are shown in Figure 2. Figure 8 It can be determined that riboflavin is covalently coupled to polyallylamine, where the ratio of flavin to allylamine repeating units is 1 / 80.

[0068] 3. Preparation of flavin mononucleotide / polyallylamine blends

[0069] Considering the possible electrostatic interaction between flavin mononucleotide and polyallylamine, they were blended as a control group. Flavin mononucleotide and polyallylamine were mixed in water to ensure that the molar ratio of flavin to allylamine repeating units was 1 / 80, and then freeze-dried, and the freeze-dried powder sample was stored at 2-8°C in the dark.

[0070] 4. Catalyze NADH to convert NAD + Comparative test of the speed

[0071] (1) using ultrapure water to dissolve the flavin / polyallylamine conjugate to obtain a solution with a flavin concentration of 100 μM, and storing the solution in the dark at room temperature after the preparation;

[0072] (2) Dissolve NADH in ultrapure water to obtain a 10 mM solution. After preparation, store the solution at 2-8°C.

[0073] (3) Take 1 μl of the solution of (1) and (2) respectively and add them to a micro-cuvette containing 98 μl of 50 mM phosphate buffer, mix them quickly and evenly, measure the change of absorbance at 340 nm, and record the flavin / polyallylamine conjugate as a catalyst to catalyze the conversion of NADH to NAD + The flavin mononucleotide / polyallylamine blend was used as the control group.

[0074] Fig. 9 The activity comparison chart of flavin / polyallylamine conjugate and blend is shown in Figure 2. Fig. 9The activity data show that the rate of NADH oxidation catalyzed by the flavin / polyallylamine conjugate is 0.21 μM s -1 , which is significantly higher than that of the flavin mononucleotide / polyallylamine blend catalyst (the rate is 0.025 μM s -1 ).

[0075] Example 3 Preparation of flavin / poly((α-lysine)5-(arginine) 15 )Conjugate

[0076] 1. Riboflavin is oxidized by hypochlorous acid to generate aldehyde flavin molecules, and the product is separated and purified to obtain a yellow powder with a yield of about 70%; the aldehyde group is then reduced to generate monohydroxy flavin with a yield of 90%; N,N'-carbonyldiimidazole is used as a coupling activation reagent to chemically connect the hydroxyl group of riboflavin and the amino group of the polymer, and then purified by dialysis to obtain flavin / poly((α-lysine)5-(arginine) 15 ) conjugate, the yield is about 52%, the structural formula is:

[0077]

[0078] 2. Flavin / Poly((α-lysine)5-(arginine) 15 ) Chemical structure characterization of conjugates

[0079] The structure was characterized by UV-visible spectroscopy, confirming that riboflavin was covalently coupled to poly((α-lysine)5-(arginine) 15 ), where the ratio of flavin group to lysine is 1 / 1. Fig.10 Flavin / poly((α-lysine)5-(arginine) 15 ) UV-visible absorption spectra of the conjugate and flavin mononucleotide.

[0080] 3. Preparation of flavin mononucleotide / poly((α-lysine)5-(arginine) 15 ) Blend

[0081] Considering that flavin mononucleotide and poly((α-lysine)5-(arginine) 15 ) possible electrostatic interaction, and they were mixed as a control group. 15 ) are mixed in water to ensure that the molar ratio of flavin to lysine repeating units is 1 / 1, and then freeze-dried. The freeze-dried powder sample is stored in a dark environment at 2-8°C to catalyze the conversion of NADH to NAD + Comparative test of the speed.

[0082] 4. Catalyze the conversion of NADH to NAD +Comparative test of the speed

[0083] (1) Dissolve flavin / poly((α-lysine)5-(arginine) in ultrapure water 15 ) coupling compound catalyst, obtain a solution with a flavin concentration of 100 μM, and store it in the dark at room temperature after preparation;

[0084] (2) Dissolve NADH in ultrapure water to obtain a 10 mM solution. After preparation, store the solution at 2-8°C.

[0085] (3) Take 1 μl of the solution obtained in (1) and (2) respectively and add them to a micro-cuvette containing 98 μl of 50 mM phosphate buffer, mix them quickly and evenly, measure the change in absorbance at 340 nm, and record the conversion of NADH to NAD catalyzed by the catalyst. + rate and conversion rate.

[0086] Fig.11 Flavin mononucleotide / poly((α-lysine)5-(arginine) 15 ) conjugates and blends, in which poly((α-lysine)5-(arginine) 15 ) is referred to as poly((α-K)5-(Y) 15 ).from Fig.11 The activity data show that flavin / poly((α-lysine)5-(arginine) 15 ) The rate of NADH oxidation activity catalyzed by the conjugate (0.14 μM s -1 ) was significantly higher than flavin mononucleotide / poly((α-lysine)5-(arginine) 15 ) blend catalyst (0.019 μM s -1 ).

[0087] Example 4

[0088] 1. Preparation of flavin / chitosan conjugate

[0089] Using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as an acid group activator, flavin mononucleotide and the amino group of chitosan (deacetylation degree ≥ 95%; viscosity 100-200mPa.s) are chemically linked, and after purification by dialysis, flavin / chitosan catalyst is obtained with a yield of about 65%. The structural formula of flavin / chitosan conjugate is:

[0090] Where n=730~1500.

[0091] 2. Chemical structure characterization of flavin / chitosan conjugates

[0092] Its structure was characterized using UV-visible spectroscopy. Fig.12 The UV-visible absorption spectra of flavin / chitosan conjugate and flavin mononucleotide are shown in Figure 2. Fig.12 It can be determined that riboflavin is covalently grafted to chitosan, where the ratio of riboflavin to amino group is 1 / 30.

[0093] 3. Preparation of flavin / chitosan blend catalyst

[0094] The flavin mononucleotide and chitosan were mixed in water to ensure that the molar ratio of flavin group to chitosan repeating unit was 1 / 30, and then freeze-dried. The freeze-dried powder sample was stored at 2-8°C in the dark, and then catalyzed to convert NADH to NAD + Comparative test of the speed.

[0095] 4. Catalyze the conversion of NADH to NAD + Comparative test of the speed

[0096] (1) using ultrapure water to dissolve the catalyst to obtain a solution with a flavin concentration of 100 μM, and storing it in the dark at room temperature after the preparation is completed;

[0097] (2) Dissolve NADH in ultrapure water to obtain a 10 mM solution. After preparation, store the solution at 2-8°C.

[0098] (3) Take 1 μl of the solution obtained in (1) and (2) respectively and add them to a micro-cuvette containing 98 μl of 50 mM phosphate buffer, mix them quickly and evenly, measure the change in absorbance at 340 nm, and record the conversion of NADH to NAD catalyzed by the catalyst. + rate and conversion rate.

[0099] Fig.13 The activity comparison chart of flavin / chitosan conjugate and blend is shown in Figure 2. Fig.13 As can be seen from the figure, the rate of catalytic NADH oxidation activity of flavin / chitosan conjugate (0.0120 μM s -1 ) was higher than that of the flavin mononucleotide / chitosan blend catalyst (0.0043 μM s -1 ).

[0100] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. The polymer coupled with flavin groups catalyzes the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD + The application is characterized by: In a multi-component solution, the polymer coupled with flavin groups catalyzes the conversion of NADH to NAD + The conversion rate is 0.007 μM / s to 0.85 μM / s, and the conversion efficiency is 20% to 100%. The polymer coupled with flavin groups is a product of flavin coupled with a polymer containing a cationic group side group through a covalent bond, and the molar ratio of the flavin group in the product molecule to the cationic group in the polymer is 1:1 to 1:

100. The polymer is composed of repeating units containing cationic groups and other repeating units, wherein the molar fraction of the repeating units containing cationic groups is 5% to 100%.

2. The flavin group-coupled polymer according to claim 1 catalyzes the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD + The application is characterized by: The polymer coupled with flavin groups catalyzes the conversion of NADH into NAD + At the same time, it can also reduce the oxidized coenzyme Q0 and coenzyme Q10.

3. The flavin group-coupled polymer according to claim 1 catalyzes the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD + The application is characterized by: The cationic group is a protonated amine, a guanidine group, a quaternary ammonium salt, a protonated pyridine, a protonated imidazole, or a triphenylphosphine cation.

4. The flavin group-coupled polymer according to claim 1 catalyzes the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD + The application is characterized by: The polymer containing cationic side groups is polypeptide, polyallylamine or chitosan.

5. The flavin group-coupled polymer according to claim 1 catalyzes the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD + The application is characterized in that The polymer coupled with flavin group is flavin / polyallylamine coupling compound, and the structural formula is Wherein, n = 800-900; or flavin / poly((glutamine) 10 -(α-Lysine) 10 ) conjugate, the structural formula is: Or flavin / chitosan conjugate, the structural formula is: Wherein, n = 730 ~ 1500; or flavin / poly((α-lysine)5-(arginine) 15 ) conjugate, the structural formula is:

6. The flavin group-coupled polymer according to claim 1 catalyzes the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD + The application is characterized in that The multi-component solution is a phosphate buffer solution.

7. The flavin group-coupled polymer according to claim 1 catalyzes the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD + The application is characterized in that The solution of multiple components is uric acid or glutathione or ascorbic acid or glutathione / uric acid mixture solution.

8. The flavin group-coupled polymer according to claim 1 catalyzes the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD + The application is characterized in that The solution of multiple components is a NaCl solution.

9. The flavin group-coupled polymer according to claim 1 catalyzes the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD + The application is characterized in that The solution of multiple components is a culture medium.

10. The flavin group-coupled polymer according to claim 1 catalyzes the conversion of reduced NADH of nicotinamide adenine dinucleotide into oxidized NAD + The application is characterized in that The culture medium comprises 90% MEM+10% FBS+1% PS, wherein the FBS is derived from fetal bovine serum and the PS contains penicillin-streptomycin dual antibodies.

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