Ec riboflavin kinase mutants and uses thereof

Abstract

The application discloses an EC riboflavin kinase mutant and application thereof, and belongs to the technical field of riboflavin kinase.The amino acid sequence of the EC riboflavin kinase mutant is shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.The EC riboflavin kinase mutant with high activity, high alkali resistance, high selectivity and suitable for industrial application is obtained by site-directed mutagenesis and screening of the original riboflavin kinase, and the mutant can be used for preparing FMN and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of riboflavin kinase technology, specifically relating to an EC riboflavin kinase mutant and its applications. Background Technology

[0002] Commercially available flavin mononucleotides (CAS No.: 146-17-8) are usually found in sodium salt form (CAS No.: 130-40-5), namely sodium riboflavin 5'-phosphate or sodium riboflavin phosphate. The main methods for industrial-scale production of FMN (flavin mononucleotides) both domestically and internationally are chemical methods, whole-cell / microbial fermentation methods, and enzymatic methods.

[0003] There are two main synthetic routes using chemical synthesis: one is to react riboflavin with partially hydrolyzed phosphorus oxychloride (POCl3) in an organic solvent to generate FMN, then hydrolyze excess POCl3, neutralize with NaOH solution, and filter to obtain sodium riboflavin 5'-phosphate; the other is to react riboflavin with POCl3 in an organic solvent to generate cyclic riboflavin 4',5'-phosphochloridate, then hydrolyze the phosphoryl compound and excess POCl3, neutralize with NaOH solution, and filter to obtain sodium riboflavin 5'-phosphate. The chemical synthesis process is simple, but the resulting sodium riboflavin phosphate product consists of sodium 3'-riboflavin phosphate, sodium 4'-riboflavin phosphate, sodium 5'-riboflavin phosphate (common name: sodium riboflavin phosphate), riboflavin diphosphate (sodium 3'4'-riboflavin phosphate, sodium 3'5'-riboflavin phosphate, sodium 4'5'-riboflavin phosphate), free riboflavin, and other impurities. In particular, the content of related substances riboflavin diphosphate and free riboflavin is high, and the presence of many impurities severely affects the purity of sodium 5'-riboflavin phosphate.

[0004] Microbial fermentation synthesizes riboflavin and FMN de novo from inexpensive raw materials such as glucose through direct metabolism by microorganisms. The highest reported yield (fermentation) of high-yield riboflavin mononucleotide (FMN) E. coli strains is 1537.2 mg / L. Due to the complexity of the synthesis pathways of riboflavin and FMN, the current production capacity of FMN by microbial fermentation cannot meet the needs of commercial production, thus limiting the application and market size of FMN in the pharmaceutical, food, and other fields.

[0005] Enzymatic methods yield FMN by catalyzing the reaction of riboflavin with adenosine triphosphate (ATP) via riboflavin kinase. Compared to chemical and whole-cell / microbial fermentation methods, enzymatic methods offer milder reaction conditions and higher selectivity, but they suffer from drawbacks such as low reactivity and poor alkali resistance of proteases. Furthermore, the selectivity needs to be further improved. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides an EC riboflavin kinase mutant and its applications. This invention obtains an EC riboflavin kinase mutant with high activity, high alkali resistance, high selectivity, and suitability for industrial applications through site-directed mutagenesis and screening of the original riboflavin kinase. This mutant has broad application prospects in the preparation of FMN.

[0007] The technical solution adopted by this invention to solve its technical problem is:

[0008] The present invention provides an EC riboflavin kinase mutant, the amino acid sequence of which is shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0009] This invention provides an EC riboflavin kinase mutant nucleotide sequence, which is obtained by mutating N9D / N24K / Q58E / C92S / D125R / D136K / Q166A, N24K / Q58E / C92S / A100I / D125R / D136K / Q166A, N24K / N47D / Q58E / C92S / A100I / D125R / E134I / Q166A, or N24K / N47D / Q58E / C85A / D125R / E134I / Q166A using SEQ ID NO: 1 as a template; and is used to express the above-mentioned EC riboflavin kinase mutant. Wherein, N9D indicates that the nucleotide corresponding to amino acid N at position 9 of the template is mutated to the nucleotide corresponding to amino acid D.

[0010] The present invention provides an EC riboflavin kinase mutant plasmid, wherein the EC riboflavin kinase mutant plasmid contains the above-mentioned EC riboflavin kinase mutant nucleotide sequence.

[0011] This invention provides an EC riboflavin kinase mutant bacterium, wherein the EC riboflavin kinase mutant bacterium contains the aforementioned EC riboflavin kinase mutant nucleotide sequence or the aforementioned EC riboflavin kinase mutant plasmid. It is used for expressing the EC riboflavin kinase mutant.

[0012] Preferably, the bacteria are Escherichia coli Rosetta strain, Escherichia coli DH5α strain, or Escherichia coli Top10 strain.

[0013] The present invention provides an EC riboflavin kinase mutant immobilized enzyme, wherein the EC riboflavin kinase mutant immobilized enzyme includes the above-mentioned EC riboflavin kinase mutant.

[0014] Preferably, the EC riboflavin kinase mutant immobilized enzyme further includes a solid-phase carrier.

[0015] More preferably, the EC riboflavin kinase mutant and the solid-phase carrier are covalently linked.

[0016] This invention provides a method for preparing flavin mononucleotide, comprising the following steps:

[0017] The above-mentioned EC riboflavin kinase mutant or the above-mentioned EC riboflavin kinase mutant immobilized enzyme is added to a mixture containing at least one of adenosine triphosphate and adenosine triphosphate salt and riboflavin to obtain flavin mononucleotide.

[0018] Preferably, the EC riboflavin kinase mutant is obtained by expression of the above-mentioned EC riboflavin kinase mutant bacteria; the EC riboflavin kinase mutant immobilized enzyme is obtained by covalently linking the EC riboflavin kinase mutant to a solid-phase carrier.

[0019] Preferably, the amount of EC riboflavin kinase mutant added is 1-5 g / L, and the amount of EC riboflavin kinase mutant immobilized enzyme added is 2-10 g / L.

[0020] More preferably, the amount of EC riboflavin kinase mutant added is 1.5 g / L, and the amount of EC riboflavin kinase mutant immobilized enzyme added is 4.0 g / L.

[0021] Preferably, the concentration of riboflavin in the mixture containing at least one of adenosine triphosphate and adenosine triphosphate salt and riboflavin is 5-50 g / L, the total concentration of adenosine triphosphate and adenosine triphosphate salt is 50-200 mM, and the pH is 7.0-10.0.

[0022] Preferably, the reaction temperature is 35-40℃ and the reaction time is 10min-7h.

[0023] Preferably, after the reaction is completed, the EC riboflavin kinase mutant immobilized enzyme is recovered and reused.

[0024] Further preferred, the EC riboflavin kinase mutant immobilized enzyme was recovered more than 20 times. Specifically, the EC riboflavin kinase mutant immobilized enzyme retained more than 98% of its enzyme activity after 20 recoveries.

[0025] The beneficial effects of this invention are:

[0026] (1) The enzyme activity of the EC riboflavin kinase mutant after mutation in this invention is significantly increased, and the enzyme activity is more than doubled;

[0027] (2) The alkali resistance of the EC riboflavin kinase mutant after mutation in this invention is significantly improved, and it still retains more than 90% of the enzyme activity after 1 hour of alkaline heating treatment;

[0028] (3) The mutated EC riboflavin kinase mutant of the present invention can catalyze the reaction of riboflavin at a concentration of 100 mM or higher to generate FMN, with a molar conversion rate of substrate riboflavin of over 93%;

[0029] (4) The immobilized enzyme of the present invention can be recovered and re-reacted multiple times, reducing the production cost of the product, while reducing protein impurities in the product and shortening the purification cycle of FMN.

[0030] (5) Compared with chemical synthesis, the method for preparing flavin mononucleotides in this invention does not require the addition of organic reagents, has high product specificity, and can generate high-purity FMN. Compared with microbial fermentation, the product concentration of enzyme-catalyzed reaction is high, which reduces the cost of product purification. Attached Figure Description

[0031] Figure 1 The liquid chromatogram of an FMN sample purchased from the market;

[0032] Figure 2 The liquid chromatogram of the riboflavin solution is shown.

[0033] Figure 3 This is the liquid chromatogram before the reaction in Example 13;

[0034] Figure 4 This is the liquid chromatogram of Example 13 at the end of the reaction. Detailed Implementation

[0035] The present invention will be further described below with reference to embodiments.

[0036] The following will clearly and completely describe the concept, specific solutions, and technical effects of the present invention with reference to embodiments, so as to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. The various technical features in the present invention can be combined interactively without contradicting each other.

[0037] The specific implementation process of this invention mainly includes: PCR amplification of wild-type nucleotide sequence → sequencing verification → gene mutation → sequencing verification → enzyme activity determination → alkaline stability enzyme activity determination → enzyme catalysis experiment.

[0038] Example 1

[0039] Construction of wild-type riboflavin kinase plasmid and strain:

[0040] The riboflavin kinase nucleotide sequence of Escherichia coli was found on NCBI. The corresponding primers were designed using Primer Premier6. NotI restriction sites were added to the C-terminal primer of the riboflavin kinase nucleotide sequence and EcoRI restriction sites were added to the N-terminal primer.

[0041] Nucleic acid extract from *Escherichia coli* strain BL21 was used as a template for PCR amplification using designed primers. The amplified fragment was then double-digested with restriction endonucleases NotI and EcoRI (purchased from New England Biolabs, NEB) at 37°C for 2 h, separated by 1% agarose gel electrophoresis, and recovered by gel extraction (gel recovery kit purchased from Tiangen Biotech (Beijing) Co., Ltd.). Subsequently, it was ligated with the expression vector pET-28a(+) (General Biotech), which had undergone the same double-digestion, at 22°C for 30 min using T4 DNA ligase (purchased from New England Biolabs, NEB). The ligation solution was transformed into competent cells of the Top 10 *E. coli* strains. Single clones were selected, and plasmids were extracted for PCR and sequencing verification. The obtained EC-wildtype plasmid was sequenced, and its riboflavin kinase nucleotide sequence is shown in SEQ ID NO: 1. The EC-wildtype plasmid was transformed into the expression host strain *Rosetta* to obtain the EC-wildtype strain, and the amino acid sequence of the expressed riboflavin kinase is shown in SEQ ID NO: 2.

[0042] SEQ ID NO: 1

[0043] ;

[0044] SEQ ID NO: 2

[0045] MKLIRGIHNLSQAPQEGCVLTIGNFDGVHRGHRALLQGLQEEGRKRNLPVMVMLFEPQPLELFATDKAPARLTRLREKLRYLAECGVDYVLCVRFDRRFAALTAQNFISDLLVKHLRVKFLAVGDDFRFGAGREGDFLLLQKAGMEYGFDITSTQTFLRRWRAYQQHRRASGSCG.

[0046] Example 2

[0047] Construction of mutant plasmids and strains:

[0048] After reviewing literature and performing molecular docking through 3D modeling, the active pocket of riboflavin kinase was determined, and suitable mutation sites were screened to design corresponding primers for mutation.

[0049] (1) Selection of gene mutation sites:

[0050] Direction: To improve alkali resistance and increase enzyme activity;

[0051] The theoretical basis for screening mutation sites:

[0052] S1. Surface residue basicization: Mutate surface Asp(D) and Glu(E) to Lys(K) and Arg(R) (positively charged residues) to reduce negative charge repulsion in an alkaline environment and enhance protein stability; preferentially select residues with an exposure of >50% (such as N-terminus, C-terminus and loop region, i.e. surface loop region) to avoid mutations in the α-helix / β-sheet of the core region;

[0053] S2. Reduce unstable groups: Replace Cys(C) (easily oxidized and crosslinked) with Ser(S) / Ala(A), and replace Asn(N) / Gln(Q) (easily deamided) with Asp(D) / Glu(E) or Ala(A) to avoid covalent bond breakage under alkaline conditions;

[0054] S3. Enhance hydrogen bonding and hydrophobic interactions: Introduce hydrophobic residues such as Leu (L) and Ile (I) by mutation in the core region of the protein (mutate only 1-2 sites), or add Arg (R)-Asp (D) and Lys (K)-Glu (E) salt bridges on the surface to maintain the rigidity of the three-dimensional structure.

[0055] (2) Mutation: KOD One™ PCR Master Mix from Toyobo was used.

[0056] S1. The PCR system is shown in Table 1;

[0057] Table 1:

[0058]

[0059] S2. The PCR procedure is shown in Table 2;

[0060] Table 2:

[0061]

[0062] (3) The EC-wildtype plasmid was subjected to site-directed mutagenesis PCR using KOD One™ PCR Master Mix from Toyobo. The mutation sites were: N9D, N24K, N47D, Q58E, D66K, D66R, C85A, D88K, C92S, C92A, A100I, D125K, D126R, E134R, E134I, D136K, E146K, Q155E, and Q166A (the codons before and after mutation are shown in Table 3). A total of 1914 mutant plasmids were designed based on different combinations of mutation sites. 19 single-site mutations were performed in the first round, 167 double-site mutations were performed in the second round, and 288 strains from the previous round were selected for each subsequent round of mutation, for a total of 8 rounds of mutation. One site was mutated at a time. The plasmid template to be mutated and the primers used for mutation were amplified by PCR. The PCR product was digested with DpnI to obtain the mutant plasmid after one mutation, completing the mutation at the target site. Multiple mutations were performed according to the design to obtain the target mutant plasmid, which was directly transformed into E. coli DH5a strain. Single clones were selected, plasmids were extracted and sequenced. Plasmids that were successfully sequenced were transformed into the expression host strain Rosetta strain to obtain the corresponding EC- mutant strain.

[0063] Table 3: Codon Changes Before and After Mutation

[0064]

[0065] Example 3

[0066] Strain enzyme activity verification

[0067] Expression was induced in EC-wildtype and EC-mutant strains: 10 μl of the corresponding strain's glycerol was inoculated into 200 mL of LB medium (containing a final concentration of 50 mg / L kanamycin) and cultured at 37 °C for 3 h. The temperature was then lowered to 25 °C, and IPTG was added to a final concentration of 1 mM for an additional 16 h of culture. The cells were then collected. The cells were resuspended in 100 mM PBS (pH 7.6) at a ratio of 1 g to 4 mL (cell weight: PBS solution = 1 g: 4 mL), sonicated (150 W, 2 s / 2 s, 20 min), and centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant enzyme solution was collected.

[0068] The enzyme solution was diluted to 15 g / L after the protein concentration was determined using NanoDrop 2000. 100 μl of the diluted enzyme solution was added to 900 μl of reaction solution (a mixture of 5 g / L riboflavin, 8.5 g / L ATP-2Na, and 100 mM sodium phosphate, pH 8.0) and placed in a 37°C water bath at 100 rpm for 10 min.

[0069] The amount of enzyme required to convert riboflavin into 1 μmol FMN within 1 minute is defined as 1 U, and the results of some enzyme activity assays are shown in Table 4.

[0070] Table 4: Results of enzyme activity assays for wild-type and mutant strains

[0071]

[0072] According to the experimental results in Table 4, the enzyme activity of the enzyme solution of the EC mutant strain varied compared with that of the EC wild-type strain. Among them, the riboflavin kinase activity of EC mutant strains 1-4 was significantly increased, more than doubled. The corresponding amino acid sequences are shown in SEQ ID NO: 3-6.

[0073] SEQ ID NO: 3

[0074] MKLIRGIHDLSQAPQEGCVLTIGKFDGVHRGHRALLQGLQEEGRKRNLPVMVMLFEPEPLELFATDKAPARLTRLREKLRYLAECGVDYVLSVRFDRRFAALTAQNFISDLLVKHLRVKFLAVGRDFRFGAGREGKFLLLQKAGMEYGFDITSTQTFLRRWRAYQAHRRASGSCG;

[0075] SEQ ID NO: 4

[0076] MKLIRGIHNLSQAPQEGCVLTIGKFDGVHRGHRALLQGLQEEGRKRNLPVMVMLFEPEPLELFATDKAPARLTRLREKLRYLAECGVDYVLSVRFDRRFIALTAQNFISDLLVKHLRVKFLAVGRDFRFGAGREGKFLLLQKAGMEYGFDITSTQTFLRRWRAYQAHRRASGSCG;

[0077] SEQ ID NO: 5

[0078] MKLIRGIHNLSQAPQEGCVLTIGKFDGVHRGHRALLQGLQEEGRKRDLPVMVMLFEPEPLELFATDKAPARLTRLREKLRYLAECGVDYVLSVRFDRRFIALTAQNFISDLLVKHLRVKFLAVGRDFRFGAGRIGDFLLLQKAGMEYGFDITSTQTFLRRWRAYQAHRRASGSCG;

[0079] SEQ ID NO: 6

[0080] MKLIRGIHNLSQAPQEGCVLTIGKFDGVHRGHRALLQGLQEEGRKRDLPVMVMLFEPEPLELFATDKAPARLTRLREKLRYLAEAGVDYVLCVRFDRRFAALTAQNFISDLLVKHLRVKFLAVGRDFRFGAGRIGDFLLLQKAGMEYGFDITSTQTFLRRWRAYQAHRRASGSCG.

[0081] Example 4

[0082] Alkali resistance verification

[0083] Riboflavin has increased solubility under alkaline conditions. Increasing the substrate concentration can improve the catalytic efficiency of riboflavin kinase and promote the formation of FMN.

[0084] Alkaline treatment of enzyme solution: Take 1 mL of the undiluted supernatant enzyme solution obtained in Example 3, add 20 µL of sodium hydroxide (4 M), and place in a water bath at 37 °C for 1 h.

[0085] The treated enzyme solution was diluted to 15 g / L after the protein concentration was determined using NanoDrop 2000. 100 μl of the diluted enzyme solution was added to 900 μl of reaction solution (a mixture of 5 g / L riboflavin, 8.5 g / L ATP-2Na, and 100 mM sodium phosphate, pH 8.0) and placed in a 37°C water bath at 100 rpm for 10 min.

[0086] The amount of enzyme required to convert 1 μmol of riboflavin to 1 μmol of FMN within 1 minute is defined as 1 U. The enzyme activity assay results are shown in Table 5.

[0087] Table 5: Enzyme activity assay results of wild-type and mutant strains after alkaline heating treatment

[0088]

[0089] According to the experimental results in Table 5, the riboflavin kinase of EC-mutant strains 1-4 can still retain more than 90% of its enzyme activity after 1 hour of alkaline heating treatment, among which EC-mutant strains 1 and 3 have better alkali resistance.

[0090] Example 5

[0091] Enzymatic reaction of EC-wild-type strain under pH 8.0 conditions

[0092] The enzyme solution of the EC-wildtype strain prepared in Example 3 was diluted to 15 g / L after the protein concentration was determined using a NanoDrop 2000. 5 g of riboflavin (132.8 mM) and 8.5 g of ATP-2Na (154.3 mM) were dissolved in 90 mL of 100 mM pH 8.0 sodium phosphate buffer. After the substrate dissolved, its auxiliary ions (60 mM magnesium chloride, 30 mM ammonium sulfate) and 10 mL of enzyme solution were added. The reaction was carried out in a 37°C water bath, with the pH controlled within the range of 8.0 ± 0.5, and mechanically stirred at 200 rpm for 7 h. The reaction was then terminated. High-performance liquid chromatography (HPLC) analysis showed that the riboflavin substrate was completely consumed, and the molar concentration of FMN was 121.62 mM, indicating a molar yield of 91.58%.

[0093] Example 6

[0094] Enzymatic reaction of EC-mutant strain 3 under pH 8.0 conditions

[0095] The enzyme solution of EC-mutant strain 3 prepared in Example 3 was diluted to 15 g / L after the protein concentration was determined by NanoDrop 2000. 5 g of riboflavin and 8.5 g of ATP-2Na were dissolved in 90 mL of 100 mM pH 8.0 sodium phosphate buffer. After the substrate dissolved, its auxiliary ions (60 mM magnesium chloride, 30 mM ammonium sulfate) and 10 mL of enzyme solution were added. The reaction was carried out in a 37°C water bath, with the pH controlled within the range of 8.0 ± 0.5, and mechanically stirred at 200 rpm for 5 h. The reaction was then terminated. High-performance liquid chromatography (HPLC) showed that the substrate riboflavin was completely consumed, and the molar concentration of FMN was 124.33 mM, indicating a molar yield of 93.62%.

[0096] Example 7

[0097] Enzymatic reaction of EC-wild-type strain under pH 10.0 conditions

[0098] The enzyme solution of the EC-wildtype strain prepared in Example 3 was diluted to 15 g / L after the protein concentration was determined using a NanoDrop 2000. 5 g of riboflavin (132.8 mM) and 8.5 g of ATP-2Na (154.3 mM) were dissolved in 90 mL of 100 mM pH 10.0 sodium phosphate buffer. After the substrate dissolved, its auxiliary ions (60 mM magnesium chloride, 30 mM ammonium sulfate) and 10 mL of enzyme solution were added. The reaction was carried out in a 37°C water bath, with the pH controlled within the range of 10.0 ± 0.5, and mechanically stirred at 200 rpm for 9 h. The reaction was then terminated. High-performance liquid chromatography (HPLC) analysis showed that the riboflavin substrate was not completely consumed, and the molar concentration of FMN was 92.46 mM, indicating a molar yield of 69.62%.

[0099] Example 8

[0100] Enzymatic reaction of EC-mutant strain 3 under pH 10.0 conditions

[0101] The enzyme solution of EC-mutant strain 3 prepared in Example 3 was diluted to 15 g / L after the protein concentration was determined by NanoDrop 2000. 5 g of riboflavin and 8.5 g of ATP-2Na were dissolved in 90 mL of 100 mM pH 10.0 sodium phosphate buffer. After the substrate dissolved, its auxiliary ions (60 mM magnesium chloride, 30 mM ammonium sulfate) and 10 mL of enzyme solution were added. The reaction was carried out in a 37°C water bath, with the pH controlled within the range of 10.0 ± 0.5, and mechanically stirred at 200 rpm for 2.5 h. The reaction was then terminated. High-performance liquid chromatography (HPLC) showed that the riboflavin substrate was completely consumed, and the molar concentration of FMN was 129.87 mM, indicating a molar yield of 97.79%.

[0102] Example 9

[0103] Small-pot fermentation of riboflavin kinase

[0104] 10 μl of the EC-mutant strain 3 glycerol strain constructed in Example 2 was inoculated into 200 mL of LB medium (containing 50 mg / L kanamycin) and cultured at 37 °C for 16 h. Then, 200 mL / 2 L (1.0%) of the medium was inoculated into the fermentation medium and cultured at 37 °C. The pH of the fermentation culture was maintained at around 6.8. Feeding was started after 2 h of fermentation, and dissolved oxygen was maintained at around 20%. When the OD600 reached 10, the temperature was lowered to the induction temperature of 20 °C, and 0.1 mM IPTG was added. After induction, fermentation continued for 15 h. The cells were collected by centrifugation at 6000 rpm and then suspended in 100 mM pH 6.0 sodium phosphate buffer. The cells were sonicated (150 W, 2 s / 2 s, 20 min) and centrifuged at 4 °C and 12000 rpm for 20 min. The supernatant enzyme solution was collected.

[0105] Example 10

[0106] His-tagged purification and immobilization of riboflavin kinase

[0107] The enzyme solution prepared in Example 9 was adjusted to pH 7.5 with 1M sodium hydroxide solution, and NiSeplife FF affinity chromatography medium was added at a ratio of 1g / 30mg riboflavin kinase. The mixture was stirred at 200rpm for 8 hours at room temperature, and the NiSeplife FF medium adsorbed with riboflavin kinase was collected. 200mM imidazole solution (pH 8.3) was added at a mass-to-volume ratio of 1g:2ml, and the mixture was stirred at 200rpm for 3 hours at room temperature, and the eluent was collected. Dipotassium hydrogen phosphate was added to the eluent to a concentration of 0.2M, and LX-1000EP solid-phase carrier was added at a ratio of 1g / 50mg riboflavin kinase. The mixture was stirred at 200rpm for 16 hours at room temperature. The immobilized enzyme was collected by centrifugation at 500rpm using a plate centrifuge and washed with pure water.

[0108] Example 11

[0109] Enzyme activity verification of immobilized enzymes

[0110] 5 g of riboflavin, 8.5 g of ATP-2Na, and their auxiliary ions (60 mM magnesium chloride and 30 mM ammonium sulfate) were dissolved in 100 mL of 100 mM pH 10.0 sodium phosphate buffer. After the substrate dissolved, the immobilized enzyme prepared in Example 10 was added at a protein concentration of 4 g / L. The reaction was carried out in a 37°C water bath, with the pH controlled within the range of 10.0 ± 0.5, and mechanically stirred at 200 rpm for 3 h. After the reaction, the riboflavin substrate was completely consumed and the molar concentration of FMN was 127 mM, i.e., the molar yield was 95.63%. The immobilized enzyme was recovered by filtration through a 500-mesh filter cloth.

[0111] Example 12

[0112] Application experiment of immobilized riboflavin kinase

[0113] 5 g of riboflavin, 8.5 g of ATP-2Na, and their auxiliary ions (60 mM magnesium chloride and 30 mM ammonium sulfate) were dissolved in 100 mL of 100 mM pH 10.0 sodium phosphate buffer. After the substrate dissolved, the immobilized enzyme recovered in Example 11 was added. The reaction was carried out in a 37°C water bath, with the pH controlled within the range of 10.0 ± 0.5. The mixture was mechanically stirred at 200 rpm for 3 h, and the immobilized enzyme was recovered for reuse. The molar concentration of FMN was determined by high performance liquid chromatography (HPLC), and the enzyme activity retention rate was calculated by comparing it with the FMN concentration at the end of the reaction in Example 11.

[0114] The above experiment was repeated 20 times, and the results are shown in Table 6.

[0115] Table 6:

[0116]

[0117] According to the experimental results in Table 6, the immobilized riboflavin kinase retained 98% of its enzyme activity after 20 applications.

[0118] Example 13

[0119] Scale-up experiment

[0120] 500 g of riboflavin, 850 g of ATP-2Na, and its auxiliary ions (60 mM magnesium chloride and 30 mM ammonium sulfate) were dissolved in 10 L of 100 mM pH 10.0 sodium phosphate buffer. After the substrate dissolved, the immobilized enzyme prepared in Example 10 was added at a protein concentration of 4 g / L. The reaction was carried out in a 37°C water bath, with the pH controlled within the range of 10.0 ± 0.5, and mechanically stirred at 200 rpm for 2.5 h. After the reaction, the riboflavin substrate was completely consumed, and the molar concentration of FMN was 128.63 mM, i.e., the molar yield was 96.86%. The immobilized enzyme was recovered by filtration. After purification processes such as column purification, membrane filtration, and crystallization, 503.65 g of riboflavin mononucleotide with a purity of 95.78% was obtained.

[0121] The liquid chromatogram of the commercially available FMN sample (prepared by chemical method) is shown below. Figure 1 Sharpening time: 15.8 minutes; Purity: 65.15%;

[0122] The liquid chromatogram of the riboflavin solution is shown below. Figure 2 16.6min out of the game;

[0123] The liquid chromatogram before the reaction is shown in the figure. Figure 3 Riboflavin corresponds to a peak at 16.5 min, and ATP corresponds to a peak at 4.29 min.

[0124] The liquid chromatogram at the end of the reaction is shown in the figure. Figure 4 ATP corresponds to the peak at 4.29 min, ADP corresponds to the peak at 5.05 min, AMP corresponds to the peak at 8.07 min, and FMN corresponds to the peak at 15.89 min.

[0125] The above is a detailed description of the preferred embodiments of the present invention. However, the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. All such equivalent modifications or substitutions are included within the scope defined by the claims of the present invention.

Claims

1. An EC riboflavin kinase mutant, characterized in that, The amino acid sequence of the EC riboflavin kinase mutant is shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6.

2. A mutant nucleotide molecule of EC riboflavin kinase, characterized in that, The EC riboflavin kinase mutant nucleotide molecule is a nucleotide molecule expressing the EC riboflavin kinase mutant of claim 1.

3. An EC riboflavin kinase mutant plasmid, characterized in that, The EC riboflavin kinase mutant plasmid contains the EC riboflavin kinase mutant nucleotide molecule as described in claim 2.

4. An EC riboflavin kinase mutant bacterium, characterized in that, The EC riboflavin kinase mutant bacteria contain the EC riboflavin kinase mutant nucleotide molecule of claim 2 or the EC riboflavin kinase mutant plasmid of claim 3.

5. The EC riboflavin kinase mutant bacteria according to claim 4, characterized in that, The bacteria are Escherichia coli Rosetta strain, Escherichia coli DH5α strain, or Escherichia coli Top10 strain.

6. An immobilized enzyme containing an EC riboflavin kinase mutant, characterized in that, The EC riboflavin kinase mutant immobilized enzyme includes the EC riboflavin kinase mutant of claim 1.

7. The EC riboflavin kinase mutant immobilized enzyme according to claim 6, characterized in that, The EC riboflavin kinase mutant immobilized enzyme also includes a solid-phase carrier; The EC riboflavin kinase mutant and the solid-phase vector are covalently linked.

8. A method for preparing flavin mononucleotide, characterized in that, Includes the following steps: The EC riboflavin kinase mutant of claim 1 or the EC riboflavin kinase mutant immobilized enzyme of any one of claims 6-7 is added to a mixture containing at least one of adenosine triphosphate and adenosine triphosphate salt and riboflavin to obtain flavin mononucleotide.

9. The method for preparing flavin mononucleotide according to claim 8, characterized in that, The amount of EC riboflavin kinase mutant added is 1-5 g / L, and the amount of EC riboflavin kinase mutant immobilized enzyme added is 2-10 g / L; The concentration of riboflavin in the mixture containing at least one of adenosine triphosphate and adenosine triphosphate salt and riboflavin is 5-50 g / L, the total concentration of adenosine triphosphate and adenosine triphosphate salt is 50-200 mM, and the pH is 7.0-10.

0. The reaction temperature is 35-40℃, and the reaction time is 10 min-7 h.

10. The method for preparing flavin mononucleotide according to claim 8, characterized in that, After the reaction is completed, the EC riboflavin kinase mutant immobilized enzyme is recovered and reused; the EC riboflavin kinase mutant immobilized enzyme is recovered 20 times and still retains more than 98% of its enzyme activity.

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