Flavin adenine dinucleotide reductase mutant and its application

Abstract

The present invention discloses a flavin adenine dinucleotide reductase mutant and its application, which belongs to the field of biotechnology and bioenzyme. The wild-type flavin adenine dinucleotide reductase is subjected to directed evolution modification to screen out a flavin adenine dinucleotide reductase that can efficiently generate FADH2 and NAD using FAD and NADH as substrates. ＋ The present invention provides a variety of flavin adenine dinucleotide reductase mutants capable of efficiently catalyzing the regeneration of FADH2. These mutants exhibit enhanced catalytic activity compared to wild-type flavin adenine dinucleotide reductase, thereby improving the catalytic effect of reducing FAD to FADH2. This improves the production efficiency of high-value chemicals that rely on the flavin adenine dinucleotide reductase regeneration cycle system. Furthermore, the reaction conditions are mild and the reaction pH range is wide, thereby reducing production costs.

Description

Technical Field

[0001] The invention belongs to the fields of biotechnology and bioenzymes, and in particular relates to a flavin adenine dinucleotide reductase mutant and an application thereof. Background Art

[0002] FADH2-dependent monooxygenase-catalyzed reactions play a key role in natural product synthesis. Many important biosynthetic reactions require the consumption of large quantities of the reduced form of flavin adenine dinucleotide (FADH2). For example, FADH2-dependent monooxygenase-catalyzed reactions play a crucial role in natural product synthesis. These enzymes catalyze numerous oxidation reactions with chemoselectivity, regioselectivity, and stereoselectivity, participating in the synthesis of a wide range of natural products. Furthermore, FADH2 serves as a key reducing agent in the reduction of ketones, aldehydes, and imines, converting these compounds to their corresponding alcohols or amines. However, the high cost of FADH2 has limited the production of high-value chemicals that require FADH2 for catalytic reactions. Therefore, developing an efficient and economical FADH2 recycling system could improve the production efficiency of these high-value chemicals and significantly reduce production costs.

[0003] Flavin adenine dinucleotide reductase (FAD reductase) belongs to the oxidoreductase class and can catalyze the redox reaction between flavin adenine dinucleotide (FAD) and reduced form of nicotinamide adenine dinucleotide (NADH) to generate FADH2 and oxidized form of nicotinamide adenine dinucleotide (NADH). + ), plays an important role in the regeneration of FADH2. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a flavin adenine dinucleotide reductase mutant capable of efficiently regenerating FADH2, and to provide a nucleic acid encoding the mutant, a recombinant vector, a recombinant cell and a product comprising the mutant, as well as a preparation method and application of the mutant.

[0005] Technical solution: The flavin adenine dinucleotide reductase mutant of the present invention has an amino acid sequence obtained by amino acid mutation of the sequence shown in SEQ ID NO: 1, wherein the mutation is at least one of N68G, R106P, R169A, and A172L.

[0006] N68G, R106P, R169A, and A172L all use the standard substitution notation of the standard single-letter amino acid code. For example, N68G indicates that the asparagine (N) at position 68 of the N-terminus of SEQ ID NO: 1 is replaced by glycine (G); R106P / R169A indicates that the arginine (R) at position 106 of the N-terminus of SEQ ID NO: 1 is replaced by proline (P), and the arginine (R) at position 169 is replaced by alanine (A).

[0007] The flavin adenine dinucleotide reductase mutants are preferably R169A, N68G, R106P, A172L, N68G / R106P / R169A / A172L, N68G / R106P / R169A, R106P / R169A, N68G / R169A / A172L, R169A / A172L, R106P / R169A / A172L, N68G / R106P, N68G / R106P / A172L, R106P / A172L, and their amino acid sequences are SEQ ID NOs: 2-14, respectively.

[0008] The present invention utilizes the sequence and structural information of flavin adenine dinucleotide reductase that has been publicly reported, and through non-redundant searches in databases such as NCBI, screens out some potential enzyme genes based on principles such as protein structure similarity, conserved site analysis, and host source diversity. These genes are functionally expressed in an E. coli expression system and then purified to obtain purified flavin adenine dinucleotide reductase mutants. Specifically, a semi-rational design is used to perform directed evolution modification on the above-mentioned flavin adenine dinucleotide reductase gene, obtaining a catalytic reaction using FAD and NADH as substrates to produce FADH2 and NAD. ＋ A flavin adenine dinucleotide reductase mutant with high catalytic activity.

[0009] The present invention also provides a nucleic acid encoding the flavin adenine dinucleotide reductase mutant, which can be obtained by base mutation of the sequence shown in SEQ ID NO: 15.

[0010] The present invention also provides a recombinant vector comprising the nucleic acid, wherein the recombinant vector comprises an expression vector.

[0011] The present invention also provides a recombinant cell comprising the recombinant vector. The recombinant cell is a host cell transformed or transfected with the expression vector.

[0012] The present invention also provides a method for preparing the flavin adenine dinucleotide reductase mutant, which comprises constructing and culturing the recombinant cell and inducing expression of the flavin adenine dinucleotide reductase mutant.

[0013] The flavin adenine dinucleotide reductase mutant can catalyze the reduction of FAD into FADH2 in the form of cell lysate or pure enzyme solution.

[0014] Preferably, the method for constructing a recombinant cell is to use whole-plasmid PCR to mutate the wild-type flavin adenine dinucleotide reductase gene to obtain the target mutant gene and construct a recombinant vector; then, the recombinant vector is transformed into competent cells to obtain the recombinant cell.

[0015] Preferably, the primers for PCR are:

[0016] Primer name Serial number Primer sequences N68G upstream primer SEQ ID NO: 16 GCTGGTTTGCATTGGTCGTGATAGTCGC N68G downstream primer SEQ ID NO: 17 GCGACTATCACGACCAATGCAAACCAGC R106P upstream primer SEQ ID NO: 18 CGGTGGATGATCCCTTTCGCGATGTGCG R106P downstream primer SEQ ID NO: 19 CGCACATCGCGAAAGGGATCATCCACCG R169A upstream primer SEQ ID NO: 20 CTGATGTATTGGCGTGCCAGCTATGCC R169A downstream primer SEQ ID NO: 21 GGCATAGCTGGCACGCCAATACATCAG A172L upstream primer SEQ ID NO: 22 CGCAGCTATCTCCGCTGGCCTGTGG A172L downstream primer SEQ ID NO: 23 CCACAGGCCAGCGGAGATAGCTGCG R169A / A172L upstream primer SEQ ID NO: 24 GTATTGGCGTGCCAGCTATCTCCGCTGGCCTGTGG R169A / A172L downstream primer SEQ ID NO: 25 CCACAGGCCAGCGGAGATAGCTGGCACGCCAATAC .

[0017] The present invention also provides a product for producing FADH2, wherein the product comprises the flavin adenine dinucleotide reductase mutant, or the nucleic acid, or the recombinant vector, or the recombinant cell.

[0018] Preferably, the product comprises immobilized enzymes or immobilized cells produced using immobilization technology.

[0019] The present invention also provides the preparation method or the product in catalyzing FAD and NADH to generate FADH2 and NAD ＋ Application in.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0021] 1. The present invention provides a variety of flavin adenine dinucleotide reductase mutants that can efficiently catalyze the regeneration of FADH2. The flavin adenine dinucleotide reductase mutants have stronger catalytic activity than wild-type flavin adenine dinucleotide reductase, thereby improving the catalytic effect of catalyzing the reduction of FAD to FADH2, thereby improving the production efficiency of high-value chemicals that rely on the flavin adenine dinucleotide reductase regeneration cycle system and reducing production costs;

[0022] 2. The mutant of the present invention can effectively improve the regeneration efficiency of FADH2. The catalytic efficiency of the mutant R106P / A172L is 4.3 times that of the wild-type flavin adenine dinucleotide reductase.

[0023] 3. The catalytic reaction conditions of the mutant of the present invention are mild and the reaction pH range is wide, with a reaction temperature of 20-50°C and a reaction pH of 3-10. Its stability is improved compared to that of the wild-type flavin adenine dinucleotide reductase. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Reaction diagram of flavin adenine dinucleotide reductase catalyzing the reduction of FAD to FADH2;

[0025] Figure 2 This is a graph showing the relative activities of the wild-type flavin adenine dinucleotide reductase and the flavin adenine dinucleotide reductase mutant in catalyzing the regeneration of FADH2 provided in the examples of the present application;

[0026] Figure 3 This is a graph showing the relative activity of wild-type flavin adenine dinucleotide reductase and flavin adenine dinucleotide reductase mutant (R106P / A172L mutant) in catalyzing the regeneration of FADH2 at different temperatures;

[0027] Figure 4 This is a graph showing the relative activity of wild-type flavin adenine dinucleotide reductase and flavin adenine dinucleotide reductase mutant (R106P / A172L mutant) in catalyzing the regeneration of FADH2 at different pH values. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0029] Example 1 is based on the golden yellow North spore ( Kitasatospora aureofaciens The flavin adenine dinucleotide reductase of ) is the original enzyme (wild type), and the gene of this enzyme was synthesized by Genwi (Suzhou) Co., Ltd. Its amino acid sequence is shown in SEQ ID NO: 1, and its gene sequence is shown in SEQ ID NO: 15. The flavin adenine dinucleotide reductase can catalyze the reaction using FAD and NADH as substrates to produce FADH2 and NAD ＋ , the reaction process is as follows Figure 1 shown.

[0030] Example 1 Flavin adenine dinucleotide reductase mutant R169A and its preparation method

[0031] This example provides a flavin adenine dinucleotide reductase mutant R169A, whose amino acid sequence is shown in SEQ ID NO: 2, and whose preparation method is as follows:

[0032] Step 1: constructing a nucleic acid encoding the flavin adenine dinucleotide reductase mutant and an expression vector encoding the nucleic acid.

[0033] The wild-type flavin adenine dinucleotide reductase gene of Bacillus aureus was mutated by whole-plasmid PCR to obtain the target mutant gene (i.e., nucleic acid encoding the flavin adenine dinucleotide reductase mutant R169A), which was then constructed on the pET22b plasmid (i.e., the expression vector encoding the nucleic acid).

[0034] The PCR primers for the nucleic acid encoding the flavin adenine dinucleotide reductase mutant R169A are SEQ ID NO: 20 and SEQ ID NO: 21.

[0035] The PCR reaction system of the whole plasmid PCR is shown in Table 1.

[0036] Table 1 PCR reaction system

[0037] Ingredients volume 10×Buffer for KOD-Plus- 2.5 μL 2 mM dNTP 2.5 μL 25 mM MgSO4 1.5 μL DMSO 1 μL 10 pmol / μL Forward Primer 0.75 μL 10 pmol / μL Reverse Primer 0.75 μL DNA template <100 of KOD-Plus- 1 μL ddH2O up to 25 μL

[0038] The PCR reaction program of the whole plasmid PCR is shown in Table 2.

[0039] Table 2 PCR reaction schedule

[0040] Reaction temperature time 95 ℃ 3 min 95 ℃ 20 s 57 ℃ 10 s 70 ℃ 4 min 12 ℃ 10 min

[0041] In this program, "95°C 20s--57°C 10s" was cycled 32 times.

[0042] After PCR amplification of the target fragment, the amplified product was detected by 1% agarose gel electrophoresis. The results showed that the amplified product was a single band of approximately 6000 bp in size. The amplified product was purified and recovered using a DNA gel purification kit.

[0043] Finally, the pET22b plasmid of the flavin adenine dinucleotide reductase mutant R169A, namely the expression vector of the flavin adenine dinucleotide reductase mutant R169A, was obtained.

[0044] Step 2: Construct host cells for transfection of expression vectors.

[0045] Escherichia coli BL21 (DE3) strain (hereinafter referred to as E. coli BL21 (DE3)) was used as the expression host (i.e., host cell), and the sequenced pET22b plasmid was transferred into E. coli BL21(DE3), construction of recombinant mutant expression strain E. coli BL21(DE3) / pET22b-KaFAD-RED.

[0046] Specifically, the purified gene fragments were digested with EasyCut endonuclease-DpnI to remove the template, and then recombined with recombinase. E. coli DH5α competent cells were plated on the surface of LB solid medium containing 100 mg / mL ampicillin and cultured at 37°C for 14 hours. A single colony was then picked and transferred to LB liquid culture and cultured at 37°C with shaking at 220 rpm for 16 hours. After the culture was completed, a portion of the bacterial suspension was added with sterile glycerol to a final glycerol concentration of 25%. The cells were numbered and stored at 80°C until further use. This was to obtain recombinant mutant plasmid clones. E. coli DH5α / pET22b-KaFAD-RED; a portion of the bacterial suspension was centrifuged at 8,000 rpm for 3 min and the cells were collected and purified using a high-purity plasmid extraction kit. E. coli The plasmid was extracted from DH5α / pET22b-KaFAD-RED, and the correctness of the mutation site was verified by sequencing.

[0047] Finally, a recombinant mutant plasmid clone strain of the flavin adenine dinucleotide reductase mutant R169A and a corresponding recombinant mutant plasmid, namely a host cell transfected with an expression vector of the flavin adenine dinucleotide reductase mutant R169A, were obtained.

[0048] Step 3: construct a recombinant mutant protein expression strain to obtain a culture containing the flavin adenine dinucleotide reductase mutant R169A.

[0049] The successfully sequenced pET22b plasmid was transformed into E. coli BL21 (DE3) as the expression host (i.e., host cell) to construct the recombinant mutant protein expression strain E. coli BL21 (DE3) / pET22b-KaFAD-RED.

[0050] Specifically, the successfully constructed recombinant mutant plasmid was transformed into E. coli BL21 (DE3) competent cells were plated onto a plate containing ampicillin at a final concentration of 100 μg / mL, and a single colony was picked and inoculated into a culture tube containing 5 mL of LB liquid medium containing 100 μg / mL ampicillin. The culture was shaken at 37°C and 220 rpm for 16 h to obtain a recombinant mutant protein expression strain. E. coli BL21(DE3) / pET22b-KaFAD-RED. 3 mL of inoculum was transferred to 500 mL of LB medium containing 100 μg / mL ampicillin. 600 When the p-value reached about 0.6, 0.5 mL of 0.5 M IPTG (i.e., isopropyl-β-D-thiogalactopyranoside) was added to make the final concentration of IPTG 0.5 mM. The culture was induced at 18°C ​​for about 16 h to obtain a culture containing the flavin adenine dinucleotide reductase mutant R169A.

[0051] Finally, a culture containing the flavin adenine dinucleotide reductase mutant R169A was obtained.

[0052] After induction, the cells were harvested by centrifugation, resuspended in buffer, and disrupted by ultrasonication in an ice bath (2 s on, 5 s off, 30 min on), followed by centrifugation at 12,000 rpm / min for 20 min at 4°C. The supernatant was collected and filtered through a 0.22 μm water filter as a sample, and the enzyme was purified by nickel column. The molar absorptivity of the protein was calculated based on the amino acid sequence of KaFAD-RED, and the purified protein was purified by A 280 The absorbance of the protein was measured by the method, and the concentration of the protein (collectively referred to as the wild-type flavin adenine dinucleotide reductase and the flavin adenine dinucleotide reductase mutant) could be calculated.

[0053] Example 2 Relative Activities of Wild-Type Flavin Adenine Dinucleotide Reductase and Flavin Adenine Dinucleotide Reductase Mutants in Catalyzing the Regeneration of FADH2

[0054] Flavin adenine dinucleotide reductase mutants N68G, R106P, A172L, N68G / R106P / R169A / A172L, N68G / R106P / R169A, R106P / R169A, N68G / R169A / A172L, R169A / A172L, R106P / R169A / A172L, N68G / R106P, N68G / R106P / A172L, and R106P / A172L were prepared using the preparation method of Example 1. PCR primers are shown in Table 3.

[0055] Table 3 Primers for constructing mutants

[0056] Primer name Serial number Primer sequences N68G upstream primer SEQ ID NO: 16 GCTGGTTTGCATTGGTCGTGATAGTCGC N68G downstream primer SEQ ID NO: 17 GCGACTATCACGACCAATGCAAACCAGC R106P upstream primer SEQ ID NO: 18 CGGTGGATGATCCCTTTCGCGATGTGCG R106P downstream primer SEQ ID NO: 19 CGCACATCGCGAAAGGGATCATCCACCG R169A upstream primer SEQ ID NO: 20 CTGATGTATTGGCGTGCCAGCTATGCC R169A downstream primer SEQ ID NO: 21 GGCATAGCTGGCACGCCAATACATCAG A172L upstream primer SEQ ID NO: 22 CGCAGCTATCTCCGCTGGCCTGTGG A172L downstream primer SEQ ID NO: 23 CCACAGGCCAGCGGAGATAGCTGCG R169A / A172L upstream primer SEQ ID NO: 24 GTATTGGCGTGCCAGCTATCTCCGCTGGCCTGTGG R169A / A172L downstream primer SEQ ID NO: 25 CCACAGGCCAGCGGAGATAGCTGGCACGCCAATAC

[0057] The catalyst for catalyzing the reduction of FAD to FADH2 was obtained from the culture containing the flavin adenine dinucleotide reductase mutant obtained in Example 1 and this example.

[0058] When the supernatant after whole cell bacterial lysis is used as the catalyst, the reaction system is: the final concentration of 1 μmol protein or OD 600 =0.6 whole-cell bacterial lysis supernatant, 2mM NADH, 0.5mM FAD, reaction buffer 100mM potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer, pH = 7.5. The absorbance change of the substrate FAD at 445nm was measured using a microplate reader to compare relative activities.

[0059] The results are as follows Figure 2 As shown, compared with the wild-type flavin adenine dinucleotide reductase (corresponding to Figure 2 Compared with the WT in the experiment, the efficiency of the flavin adenine dinucleotide reductase mutant in catalyzing the reduction of FAD to FADH2 was significantly improved.

[0060] When pure enzyme solution was used as catalyst, the results were shown in Table 4.

[0061] Table 4 Catalytic reaction results of flavin adenine dinucleotide reductase mutants

[0062] catalyst condition concentration Relative activity Wild-type flavin adenine dinucleotide reductase Pure enzyme solution 1 μmol 100% Flavin adenine dinucleotide reductase mutant R106P / A172L Pure enzyme solution 1 μmol 431%

[0063] Depend on Figure 2 As can be seen from Table 4, the catalytic conversion rates of the mutants were all higher than those of the wild-type flavin adenine dinucleotide reductase. In particular, the flavin adenine dinucleotide reductase mutant R106P / A172L had a catalytic efficiency 4.3 times that of the wild-type flavin adenine dinucleotide reductase. In summary, the flavin adenine dinucleotide reductase mutants obtained through the examples of the present application have greater activity in catalyzing the reduction of FAD to FADH2 than the wild-type flavin adenine dinucleotide reductase, thereby improving the production efficiency of high-value chemicals that rely on the FADH2 regeneration cycle system and reducing the production cost of such high-value chemicals.

[0064] Example 3 Optimal Temperature for the Flavin Adenine Dinucleotide Reductase Mutant R106P / A172L to Catalyze FAD to FADH2

[0065] This example takes the flavin adenine dinucleotide reductase mutant R106P / A172L as an example to study the catalytic activity of the flavin adenine dinucleotide reductase mutant on NAD ＋ Preferred temperature for the catalytic reduction to NADH.

[0066] The reaction system consisted of a final concentration of 1 μmol protein, 2 mM NADH, and 0.5 mM FAD in a 100 mM potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer, pH 7.5. The reaction temperature was controlled in a water bath at 20°C, 30°C, 40°C, and 50°C. The absorbance of the FAD substrate at 445 nm was measured using a microplate reader for comparison of relative activities.

[0067] The results are as follows Figure 3 Experiments have shown that different temperatures significantly affect the catalytic activity of different mutants, with their reaction activity exhibiting a skewed normal trend with temperature. The optimal catalytic effect was observed at 30°C, while enzyme activity was affected at temperatures below or above this level, with the effect becoming more pronounced at elevated temperatures.

[0068] Example 4 Preferred method for catalyzing FAD to produce FADH2 by the flavin adenine dinucleotide reductase mutant R106P / A172L

[0069] This example takes the flavin adenine dinucleotide reductase mutant R106P / A172L as an example to study the optimal pH value of the catalytic reaction of the flavin adenine dinucleotide reductase mutant catalyzing the reduction of FAD to FADH2.

[0070] The reaction system consisted of a final concentration of 1 μmol protein, 2 mM NADH, 0.5 mM FAD, and reaction buffer. The reaction buffers consisted of 100 mM citric acid-sodium citrate buffer (Citrate Buffer) at pH 3, 4, and 5; 100 mM potassium phosphate-dihydrogen phosphate buffer (KPi) at pH 6, 7, and 8; and glycine-sodium hydroxide (Gly-NaOH) buffer at pH 9 and 10. The reaction temperature was maintained at room temperature (25°C). The absorbance of the FAD substrate at 445 nm was measured using a microplate reader for comparison of relative activities.

[0071] See the results Figure 4 The experiment showed that different buffer solutions had a significant effect on the activity of the enzyme. Under overly acidic or alkaline conditions, the enzyme activity was lost. The activity of the wild-type enzyme remained at a high level at a pH of 6-9, and the activity of the mutant remained at a high level at a pH of 5-8. Using KPi buffer as the reaction system, the activity of both the wild-type enzyme and the mutant was high at pH = 8.

[0072] Example 5 Optimal Kinetic Parameters for the Production of FADH2 from FAD Catalyzed by the Flavin Adenine Dinucleotide Reductase Mutant R106P / A172L

[0073] Based on Examples 3 and 4, this example measured the reaction rates of wild-type flavin adenine dinucleotide reductase and its mutants under different FAD concentrations, and performed a double reciprocal plot based on the reciprocal of the reaction rate and substrate concentration to calculate the kinetic parameters.

[0074] The results are shown in Table 5. Compared with the wild-type flavin adenine dinucleotide reductase, the Km of the flavin adenine dinucleotide reductase mutant R106P / A172L was reduced and r was increased, indicating that the catalytic activity of the mutant was significantly improved.

[0075] Table 5 Kinetic parameters

[0076] catalyst Km(mM) <![CDATA[r(min -1 )]]> <![CDATA[Kcat / Km(mM -1 ·min -1 )]]> Wild-type flavin adenine dinucleotide reductase 1.69±0.05 257.09±10 152.13±10 Flavin adenine dinucleotide reductase mutant R106P / A172L 0.18±0.05 505.28±10 2807.11±100

[0077] Note: Km refers to the Michaelis constant, r refers to the reaction rate, and Kcat refers to the catalytic constant.

Claims

1. A flavin adenine dinucleotide reductase mutant, the amino acid sequence of which is obtained by amino acid mutation of the sequence shown in SEQ ID NO: 1, characterized in that: The mutants are R169A, N68G, R106P, A172L, R106P / R169A, R169A / A172L, N68G / R106P or R106P / A172L.

2. A nucleic acid, characterized in that Encoding the flavin adenine dinucleotide reductase mutant according to claim 1.

3. A recombinant vector, characterized in that Comprising the nucleic acid according to claim 2.

4. A recombinant cell, characterized in that Comprising the recombinant vector according to claim 3.

5. A method for preparing the flavin adenine dinucleotide reductase mutant according to claim 1, characterized in that: The recombinant cell according to claim 4 is constructed and cultured to induce expression of the flavin adenine dinucleotide reductase mutant.

6. The preparation method according to claim 5, characterized in that The method for constructing the recombinant cell is to use whole plasmid PCR to mutate the wild-type flavin adenine dinucleotide reductase gene to obtain the target mutant gene and construct a recombinant vector; and then transform the recombinant vector into competent cells to obtain the recombinant cell.

7. The preparation method according to claim 6, characterized in that The primers for the PCR are: N68G: GCTGGTTTGCATTGGTCGTGATAGTCGC GCGACTATCACGACCAATGCAAACCAGC R106P: CGGTGGATGATCCCTTTCGCGATGTGCG CGCACATCGCGAAAGGGATCATCCACCG R169A: CTGATGTATTGGCGTGCCAGCTATGCC GGCATAGCTGGCACGCCAATACATCAG A172L: CGCAGCTATCTCCGCTGGCCTGTGG CCACAGGCCAGCGGAGATAGCTGCG R169A / A172L: GTATTGGCGTGCCAGCTATCTCCGCTGGCCTGTGG CCACAGGCCAGCGGAGATAGCTGGCACGCCAATAC.

8. A product for producing FADH2, characterized in that The product comprises the flavin adenine dinucleotide reductase mutant according to claim 1, or the nucleic acid according to claim 2, or the recombinant vector according to claim 3, or the recombinant cell according to claim 4.

9. The product according to claim 8, characterized in that The products include immobilized enzymes or immobilized cells produced by using immobilization technology.

10. The preparation method according to claim 5 or the product according to claim 8 catalyzes FAD and NADH to generate FADH2 and NAD ＋ Application in.

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