Anchoring peptide mediated fusion protein and immobilized enzyme bio-membrane reactor

Abstract

The invention belongs to the technical field of biological catalysis, and particularly relates to an anchor peptide mediated fusion protein and an immobilized enzyme biofilm reactor, the fusion protein comprises a functional enzyme protein and an anchor peptide mutant; the anchoring peptide mutant is obtained by mutating proline at the fifth position of an amino acid sequence as shown in SEQ ID No.2 into valine; the functional enzyme protein is nicotinamide ribokinase or polyphosphate kinase. The functional enzyme protein and the anchoring peptide mutant are connected by using the connecting peptide, and then the obtained fusion protein is anchored on the polypropylene hollow fiber membrane wire material, so that the obtained fusion protein has good anchoring effect and catalytic activity, the batch reaction yield stability can be obviously improved, and the preparation method is suitable for industrial production. An immobilized enzyme bio-membrane reactor is designed, and nicotinamide mononucleotide can be synthesized through biological catalysis.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to an anchored peptide-mediated fusion protein and immobilized enzyme biofilm reactor. Background Technology

[0002] Immobilized enzymes refer to the process of fixing enzymes onto water-insoluble carriers using physical or chemical methods to form insoluble derivatives. With excellent immobilization methods, enzymes or cells can be confined to a limited space without significantly reducing enzyme activity. Immobilization methods include traditional carrier adsorption, embedding, cross-linking, and covalent bonding methods, as well as novel immobilization methods. Compared to free cells, the advantages of immobilized enzymes are mainly: (1) Immobilization can alter the enzyme's performance in the catalytic process, including improving enzyme activity, stereoselectivity, and stability; (2) The operability of immobilized enzymes is improved, and enzyme recovery and reuse greatly enhance the economic feasibility of enzyme applications; (3) After the enzyme is immobilized, the catalytic system can change from homogeneous to heterogeneous, which helps in the separation of enzymes and cells, resulting in higher product purity.

[0003] Anchored peptides are short peptides that can specifically bind to a certain material. They can bind tightly to the surfaces of various natural and synthetic materials with high affinity and efficiency by relying on non-covalent multi-site interactions (such as electrostatic interactions, hydrophobic interactions, π-π stacking and hydrogen bonding). A novel enzyme immobilization method is to link enzyme proteins and anchored peptides with linking peptides. This allows enzyme proteins to bind to a certain material in a directional and specific manner through anchored peptides, while ensuring that the conformation of the active site of the enzyme is not affected, thus minimizing the impact on enzyme activity. Membrane materials, as carriers for binding, have many advantages: (1) high specific surface area and high packing density, which improves the enzyme loading and catalytic efficiency per unit volume; (2) excellent mass transfer performance, avoiding the decrease in reaction rate due to diffusion limitation; (3) reusable and with a longer lifespan; (4) strong modularity and scale-up potential, making it easy to scale up and industrialize.

[0004] Nicotinamide mononucleotide (NMN), a naturally occurring active substance in living organisms, belongs to the nucleotide class of compounds and is a precursor for the synthesis of nicotinamide adenine dinucleotide (NAD). + One of the main biological precursors of NAD. + As a key coenzyme in physiological processes such as cell metabolism, energy conversion, and DNA repair, its level is closely related to aging and metabolic diseases. Therefore, NMN shows significant application value in delaying aging and improving metabolic function. NMN mainly exists in two isomers, α and β, of which only β-NMN possesses biological activity.

[0005] Enzymatic synthesis methods mainly include the nicotinamide pathway and the nicotinamide ribokinase pathway. The latter has become the focus of current research due to the high catalytic efficiency and strong reaction specificity of its key enzyme—nicotinamide ribokinase (NRK). NRK catalyzes the phosphorylation of nicotinamide ribose (NR) to NMN in the presence of adenosine triphosphate (ATP). The addition of polyphosphoric acid kinase (PPK) can regenerate the consumed ATP, thereby saving costs. Summary of the Invention

[0006] This invention provides an anchored peptide-mediated fusion protein and immobilized enzyme biomembrane reactor, which uses anchored peptides to immobilize functional enzyme proteins on polypropylene hollow fiber membrane materials. The specific technical solution is as follows: In a first aspect, the present invention provides a fusion protein comprising a functional enzyme protein and an anchoring peptide mutant; wherein the anchoring peptide mutant is attached to the N-terminus or C-terminus of the functional enzyme protein. The anchoring peptide mutant is obtained by mutating proline at position 5 of the amino acid sequence shown in SEQ ID No. 2 to valine; The functional enzyme protein is nicotinamide ribokinase or polyphosphate kinase; The amino acid sequence of the nicotinamide ribokinase is shown in SEQ ID No. 10; the amino acid sequence of the polyphosphate kinase is shown in SEQ ID No. 12.

[0007] Furthermore, the nucleotide sequence of the anchoring peptide mutant is shown in SEQ ID No. 3, and the amino acid sequence of the anchoring peptide mutant is shown in SEQ ID No. 4.

[0008] The original anchored peptide selected in this invention contains 43 amino acid residues, with a theoretical molecular weight of 4792 Da and an isoelectric point of 7.71. It is an anionic antimicrobial peptide containing both hydrophobic and hydrophilic amino acids. The side chains of the hydrophobic amino acids form hydrophobic interactions with polymers such as polypropylene. Its amino acid sequence includes four aromatic R-group amino acids (one phenylalanine residue and three tyrosine residues) and hydrophobic amino acids such as alanine and leucine.

[0009] This invention utilizes an anchor peptide-mediated approach to achieve enzyme immobilization, and performs single-point mutations on the selected original anchor peptide to obtain an anchor peptide mutant with improved immobilization ability with the vector. Two fusion proteins prepared by combining nicotinamide ribokinase, polyphosphokinase, and the anchor peptide mutant possess both the ability of the anchor peptide mutant to bind to the vector and the ability to catalyze the conversion of substrates into products, showing promise for industrial production.

[0010] Furthermore, the nucleotide sequence of the nicotinamide ribokinase is shown in SEQ ID No. 9; the nucleotide sequence of the polyphosphate kinase is shown in SEQ ID No. 11.

[0011] Furthermore, the C-terminus of the two enzyme proteins is linked to an anchoring peptide mutant.

[0012] In a further design of the present invention, it was found that different ways of linking nicotinamide ribokinase to the anchor peptide mutant would affect the activity of the fusion protein. The fusion protein formed by linking the C-terminus of nicotinamide ribokinase to the anchor peptide mutant had better catalytic activity, reaching up to 54% of the activity of the nicotinamide ribokinase used. However, the fusion protein formed by linking the N-terminus of nicotinamide ribokinase to the anchor peptide mutant had an activity that was only about 31% to 39% of the activity of the nicotinamide ribokinase.

[0013] Furthermore, the anchoring peptide mutant is linked to nicotinamide ribokinase or polyphosphate kinase via a linker peptide.

[0014] Further, the linker peptide is an N-segment repeating sequence of a flexible linker peptide or a rigid linker peptide, where N = 1 to 3; the amino acid sequence of the flexible linker peptide is shown in SEQ ID No. 6; the amino acid sequence of the rigid linker peptide is shown in SEQ ID No. 8.

[0015] Furthermore, the nucleotide sequence of the flexible linker peptide is shown in SEQ ID No. 5; the nucleotide sequence of the rigid linker peptide is shown in SEQ ID No. 7.

[0016] In the construction of the fusion protein provided by this invention, the anchoring peptide mutant and the functional enzyme protein are selected based on the functional requirements of the fusion protein. Direct intercalation of the two proteins may create steric hindrance, affecting their activity. To avoid this, this invention uses a linker peptide to connect the anchoring peptide mutant and the functional enzyme protein. Furthermore, based on the above-mentioned design of the connection method for the two proteins, a connection method of functional enzyme protein-linker peptide-anchoring peptide mutant is selected.

[0017] Furthermore, the linker peptide is one of the following: a rigid linker peptide repeat sequence, two rigid linker peptide repeat sequences, three rigid linker peptide repeat sequences, or a flexible linker peptide repeat sequence.

[0018] Furthermore, the linker peptide is a rigid linker peptide repeat sequence.

[0019] The linker peptides used in this invention are of two types: flexible linker peptides (amino acid sequence GGGGS) and rigid linker peptides (amino acid sequence EAAAK). Flexible linker peptides, rich in glycine (Gly), have a relatively loose and flexible overall structure, making them prone to bending and conformational changes, thus giving the functional proteins at both ends of the linker peptide a high degree of freedom of movement. Rigid linker peptides, on the other hand, typically form stable α-helical secondary structures that are not easily bent. This rigidity effectively limits the relative positions and spacing of the functional proteins at both ends, fully separating different structural domains and minimizing mutual interference. Through testing, this invention found that using one of the following—one rigid linker peptide repeat sequence, two rigid linker peptide repeat sequences, three rigid linker peptide repeat sequences, or one flexible linker peptide repeat sequence—can maintain good activity of nicotinamide ribokinase and polyphosphokinase. Among these, one rigid linker peptide repeat sequence allows the anchor peptide mutant to retain better immobilization with the vector, resulting in the best preservation of the functional activity of both enzymes and the anchor peptide.

[0020] Generally, the purpose of adding linker peptides when designing fusion proteins is to prevent mutual interference between the functional domains at both ends and avoid reduced activity due to folding and entanglement. Simultaneously, linker peptides can maintain an appropriate distance between the active sites of each functional region, thereby reducing the impact of steric hindrance on their function. Appropriate types and lengths of linker peptides should be selected to achieve the above functions. In the experiments of this invention on rigid linker peptides, regarding the activity retention effect of the proteins at both ends, one rigid linker peptide repeat sequence > two rigid linker peptide repeat sequences > three rigid linker peptide repeat sequences. Since rigid linker peptides have a relatively fixed length, this indicates that the two functional proteins in the fusion protein used in this invention have better activity when they are closer together. In the experiments of this invention on flexible linker peptides, it was found that nicotinamide ribokinase exhibited the best activity with one flexible linker peptide repeat sequence.

[0021] Furthermore, the fusion protein is selected from any of the following: A1) A fusion protein containing the nicotinamide ribokinase, with the amino acid sequence shown in SEQ ID No. 14; A2) A fusion protein containing the polyphosphate kinase described in SEQ ID No. 16, with an amino acid sequence as shown.

[0022] In a second aspect, the present invention provides a gene encoding the above-mentioned fusion protein, wherein the nucleotide sequence of the gene encoding the fusion protein is selected from any of the following: B1) When the amino acid sequence of the fusion protein is SEQ ID No. 14, its encoding gene is the nucleotide sequence shown in SEQ ID No. 13; B2) When the amino acid sequence of the fusion protein is SEQ ID No. 16, its encoding gene is the nucleotide sequence shown in SEQ ID No. 15.

[0023] In a third aspect, the present invention provides a recombinant expression vector or genetically engineered bacterium for the above-mentioned encoding gene.

[0024] The present invention provides, in a fourth aspect, an immobilized enzyme comprising: Polypropylene material; and the fusion protein, as described above, immobilized on the polypropylene material.

[0025] Furthermore, the polypropylene material is in the form of hollow fiber membrane filaments.

[0026] An immobilized enzyme reactor is an enzyme reactor that uses immobilized enzymes to catalyze reactions. The immobilized enzyme exists in a closed state within a certain space, allowing for continuous reaction. The enzymes after the reaction can be recovered and reused.

[0027] An immobilized enzyme biofilm reactor is a reactor that uses membrane material as a carrier for immobilized enzymes to carry out catalytic reactions. The immobilized enzymes are fixed on the membrane, resulting in higher enzyme loading and catalytic efficiency. It is reusable, has a long lifespan, and is modular with strong potential for scale-up.

[0028] In this invention, an anchoring peptide mutant is used to anchor the fusion protein to an anchoring material, resulting in a fusion protein-polymer membrane conjugate with the function of catalytically converting nicotinamide riboside (NR) to nicotinamide mononucleotide (NMN). In actual industrial production, the sheet-like anchoring material can be other media placed in the reaction vessel or as a pipeline for liquid flow. The anchoring material provided by this invention allows for a wide range of material choices for the fusion protein, greatly facilitating its use in production. Furthermore, the fusion protein provided by this invention exhibits good anchoring performance; in multiple batches of catalytic experiments, the fusion protein maintained a good anchoring state. This advantage reduces the cost of separating the fusion protein and product in actual production. Therefore, the fusion protein and anchoring material provided by this invention have promising application prospects.

[0029] The present invention provides a method for preparing the above-mentioned immobilized enzyme in a fifth aspect, comprising the following steps: S1: Take the crude enzyme solution or purified enzyme solution of the fusion protein; S2: Take polypropylene hollow fiber membrane material, wash and dry it; S3: Mix the crude enzyme solution in S1 with the polypropylene hollow fiber membrane material in S2, and incubate them together to obtain the immobilized enzyme.

[0030] The present invention provides, in a sixth aspect, the use of the above-described immobilized enzyme, or the immobilized enzyme prepared by the above-described method, in the catalytic production of nicotinamide mononucleotide.

[0031] Furthermore, the application is carried out in a reaction system containing nicotinamide ribose, ATP, and magnesium ions.

[0032] Furthermore, the application method is selected from any of the following: C1) Using nicotinamide riboside (NR) as a substrate, an immobilized enzyme containing a fusion protein of nicotinamide ribokinase as a catalyst, and a phosphate buffer solution with a pH of 8.0 as a reaction medium, a catalytic phosphate transfer reaction is carried out using ATP as a phosphate donor, and the substrate is converted into nicotinamide mononucleotide (NMN) by obtaining phosphate groups. C2) Using nicotinamide riboside (NR) as a substrate, an immobilized enzyme containing both nicotinamide ribokinase and polyphosphate kinase as a catalyst, and a phosphate buffer solution with a pH of 8.0 as the reaction medium, a small amount of ATP is used as the phosphate donor to catalyze a phosphate group transfer reaction, and the substrate is converted into nicotinamide mononucleotide (NMN) by obtaining phosphate groups.

[0033] Furthermore, in the C2 reaction, the polyphosphate kinase and nicotinamide ribokinase are added in an equal ratio of 50 g / L.

[0034] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a novel enzyme immobilization technique using anchoring peptides, which can immobilize free nicotinamide ribokinase or a combination of nicotinamide and polyphosphate kinases on an anchoring membrane material. Furthermore, by using linking peptides to connect nicotinamide ribokinase and anchoring peptide mutants, the resulting fusion protein possesses both good anchoring effect and catalytic activity.

[0035] The PP used in this invention is a biodegradable or environmentally friendly anchoring material that is currently available.

[0036] In this invention, immobilizing the free enzyme on the anchoring material can significantly improve the reaction batch size, make the product easy to separate from the catalyst, and the anchoring material can still be used for re-incubation and binding after washing to remove impurities. This greatly reduces the cost of biocatalytic synthesis of nicotinamide mononucleotide and has good prospects for industrial application.

[0037] The multi-enzyme cascade co-immobilized biofilm reactor designed in this invention can regenerate ATP without significantly affecting the yield of nicotinamide mononucleotide, thereby reducing the amount of ATP added to the reaction system and greatly reducing production costs. Attached Figure Description

[0038] Figure 1 The image shows a comparison of fluorescence intensity between the wild-type anchor peptide and the anchor peptide mutant; where CgDef(WT) represents the original anchor peptide, CgDef(V1) represents the T36C mutant, CgDef(V2) represents the R32C mutant, CgDef(V3) represents the P5V mutant, and CgDef(V4) represents the T36V mutant.

[0039] Figure 2 This is an SDS-PAGE electrophoresis image of the cell lysate and supernatant of the recombinase containing anchor peptide mutants linked to the C-terminus of nicotinamide ribokinase by rigid and flexible linkers at different folds. In the image, the marker represents the molecular weight of the standard protein, NRK represents nicotinamide ribokinase, and in KR1D to KS3D, K represents nicotinamide ribokinase, D represents the anchor peptide mutant, R represents the rigid linker repeat sequence, S represents the flexible linker repeat sequence, and the number indicates the segment number of the linker repeat sequence.

[0040] Figure 3 This is an SDS-PAGE electrophoresis image of the cell lysate and supernatant of the recombinase containing anchor peptide mutants linked to the N-terminus of nicotinamide ribokinase by rigid and flexible linkers at different folds. In the image, the marker represents the molecular weight of the standard protein, NRK represents nicotinamide ribokinase, and in DR1K to DS3K, K represents nicotinamide ribokinase, D represents the anchor peptide mutant, R represents the rigid linker repeat sequence, S represents the flexible linker repeat sequence, and the number indicates the segment number of the linker repeat sequence.

[0041] Figure 4 This is a standard curve of protein concentration.

[0042] Figure 5 This is a standard curve of NMN concentration.

[0043] Figure 6 Immobilized enzymes were prepared by combining five fusion proteins with high free enzyme activity (KR1D, KR2D, KR3D, KS1D, and DR1K) with PP. The relative activity of each enzyme was determined by repeating the reaction six times, with the enzyme activity after the first reaction as 100%.

[0044] Figure 7 The enzymes KR1D-PP, KR1D-PVDF, KR1D-PSF, and KR1D-PTFE were immobilized by combining KR1D with four different materials. The enzyme activity of each enzyme was measured by repeating the reaction six times.

[0045] Figure 8The graph shows the NMN yield of the fusion protein-polypropylene material conjugate based on the anchored peptide mutant (KR1D-PP) in different batches at a substrate concentration of 20 mM; the NMN yield was calculated once every 2 hours of reaction during the repeated use.

[0046] Figure 9 The graph shows the NMN yield of the fusion protein-polypropylene material conjugate (KR1D-PP) based on the anchored peptide mutant at a substrate concentration of 100 mM in different batches. The NMN yield was calculated once every 6 hours of reaction during the repeated use.

[0047] Figure 10 The relative enzyme activities of KRID immobilized enzyme and free enzyme were measured after incubation at different temperatures for 2 hours, with the enzyme activities of the two enzymes without incubation treatment as 100% of the baseline.

[0048] Figure 11 The relative enzyme activities of KRID immobilized enzyme and free enzyme were measured after being stored at 4°C for different numbers of days, with the initial enzyme activities before storage as 100%.

[0049] Figure 12 The NMN yield is plotted for different batches of two fusion protein-polypropylene material conjugates (KR1D-PP-PR1D) based on anchored peptide mutants at a substrate concentration of 100 mM. The NMN yield was calculated every 6 hours during the repeated use.

[0050] Figure 13 Homologous modeling structures of fusion proteins containing nicotinamide ribokinase, and schematic diagrams of biocatalytic membrane reactors.

[0051] Figure 14 The graph shows the NMN yield of different batches of the original anchored peptide-based fusion protein-polypropylene material conjugate (KR1D-WT-PP) at a substrate concentration of 100 mM; the NMN yield was calculated once every 6 hours of reaction during the repeated use.

[0052] Figure 15 The NMN yield graphs are for different batches of the original anchored peptide-based fusion protein-polypropylene material conjugate (KR1D-WT-PP-PR1D-WT) at a substrate concentration of 100 mM. The NMN yield was calculated once every 6 hours of reaction during the repeated use. Detailed Implementation

[0053] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are exemplary and only represent a part of the embodiments of the present invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0054] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the supplier's recommended operating instructions. Unless otherwise specified, the experimental methods in this invention are conventional methods; for specific gene cloning operations, please refer to *Molecular Cloning: A Laboratory Manual* edited by J. Sambrook et al. Reagents used in upstream gene engineering operations: The one-step cloning kits used in the embodiments of this invention were all purchased from Vazyme, Beijing Qingke Biotechnology Co., Ltd.; plasmid extraction kits and DNA recovery and purification kits were purchased from Axygen Hangzhou Co., Ltd.; E. coli BL21(DE3), plasmids, etc., were purchased from Shanghai Sangon Biotech; DNA markers, low molecular weight standard proteins, agarose gel electrophoresis reagents, primer synthesis, and gene sequencing were performed by Hangzhou Qingke Biotechnology Co., Ltd. The usage methods of the above reagents are as per the product instructions.

[0055] Reagents used in downstream catalytic processes: Nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) standards, and other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. Preparation of competent cells: E. coli BL21(DE3) strain preserved in glycerol tubes was obtained from a -80℃ freezer, streaked on antibiotic-free LB agar plates, and cultured at 37℃ for 10 h to obtain single colonies; single colonies from LB agar plates were picked and inoculated into test tubes containing 10 mL of LB medium, and cultured at 37℃ and 180 rpm for 9 h; 2 mL of bacterial culture was taken from the test tube and inoculated into 100 mL of LB medium, and cultured at 37℃ and 180 rpm for OD... 600Adjust the concentration to 0.4-0.6; pre-cool the bacterial culture on ice, transfer the culture to a sterile centrifuge tube, place on ice for 10 min, centrifuge at 4℃ and 5000 rpm for 10 min; discard the supernatant, resuspend the precipitated cells in a 0.1 mol / L CaCl2 aqueous solution pre-cooled at 4℃, and place on ice for 30 min; centrifuge at 4℃ and 5000 rpm for 10 min, discard the supernatant, resuspend the precipitated cells in a 0.1 mol / L CaCl2 aqueous solution pre-cooled at 4℃ containing 15% glycerol, aliquot 100 μL of the resuspended cells into sterile 1.5 mL centrifuge tubes, and store at -80℃. Remove as needed.

[0056] Nicotinamide ribose (NR) is converted into nicotinamide mononucleotide (NMN) by nicotinamide ribokinase catalysis, as shown in the following process.

[0057]

[0058] The concentration of NMN was determined using high-performance liquid chromatography (HPLC). The analytical method was as follows: Column type: C18 column (5μm, 4.6mm × 250mm). Mobile phase: 3.48 g of anhydrous K₂HPO₄ was dissolved in ultrapure water, and the pH was adjusted to 7.0 with KH₂PO₄ before being brought to a final volume of 1L. Detection wavelength: 254nm; injection volume: 10μL; flow rate: 0.8mL / min; column oven temperature: 40°C; elution time: 3.2 minutes. The standard curve of NMN is shown below. Figure 5 As shown.

[0059] Enzyme activity unit (U) definition: Under conditions of 40℃, using nicotinamide ribose (NR) as a substrate and free enzyme or immobilized enzyme as a catalyst, the amount of enzyme required to catalyze a reaction for 3 min (free enzyme) or 10 min (immobilized enzyme) to produce 1 μmol of nicotinamide mononucleotide (NMN) per min is defined as one enzyme activity unit. The specific activity of immobilized enzyme is defined as the number of enzyme activity units (U / g) contained in one gram of immobilized enzyme.

[0060] In the following examples, CgDef(V1)(T36C), CgDef(V2)(R32C), CgDef(V3)(P5V), and CgDef(V4)(T36V) are mentioned. T36C and other descriptions refer to the mutation of threonine at position 36 of the original protein to cysteine. The specific correspondence between the amino acids and their abbreviations is as follows: threonine (T), cysteine ​​(C), arginine (R), proline (P), and valine (V).

[0061] In this invention, the nucleotide sequence of the original anchoring peptide is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2; the nucleotide sequence of the anchoring peptide mutant is shown in SEQ ID No. 3, and the amino acid sequence is shown in SEQ ID No. 4; the nucleotide sequence of the flexible linker peptide is shown in SEQ ID No. 5, and the amino acid sequence is shown in SEQ ID No. 6; the nucleotide sequence of the rigid linker peptide is shown in SEQ ID No. 7, and the amino acid sequence is shown in SEQ ID No. 8; the nucleotide sequence of nicotinamide ribokinase is shown in SEQ ID No. 9, and the amino acid sequence is shown in SEQ ID No. 10; the nucleotide sequence of polyphosphate kinase is shown in SEQ ID No. 11, and the amino acid sequence is shown in SEQ ID No. 12; the nucleotide sequence of the fusion protein containing nicotinamide ribokinase is shown in SEQ ID No. 13, and the amino acid sequence is shown in SEQ ID No. 14; the nucleotide sequence of the fusion protein containing polyphosphate kinase is shown in SEQ ID No. 15, and the amino acid sequence is shown in SEQ ID No. 16; the nucleotide sequence of the enhanced green fluorescent protein is shown in SEQ ID No. 17, and the amino acid sequence is shown in SEQ ID No. 18. As shown in No. 18, specifically: SEQ ID No. 1: GGCTTTGGCTGCCCGGAAGATGAATATGAATGCCATAATCATTGCAAAAATAGCGTGGGCTGCCGTGGCGGCTATTGCGATGCAGGCACCCTGCGTCAGCGTTGCACCTGCTATGGCTGCAATCGTAAA; SEQ ID No. 2: GFGCPEDEYECHNHCKNSVGCRGGYCDAGTLRQRCTCYGCNRK; SEQ ID No. 3: GGCTTTGGCTGCGTGGAAGATGAATATGAATGCCATAATCATTGCAAAAATAGCGTGGGCTGCCGTGGCGGCTATTGCGATGCAGGCACCCTGCGTCAGCGTTGCACCTGCTATGGCTGCAATCGTAAA; SEQ ID No.4: GFGCVEDEYECHNHCKNSVGCRGGYCDAGTLRQRCTCYGCNRK; SEQ ID No. 5: GGCGGTGGCGGCAGC; SEQ ID No.6: GGGGS; SEQ ID No.7: GAAGCGGCGGCGAAG; SEQ ID No.8: EAAAK; SEQ ID No.9: ATGACCACCACCAAGGTGAAACTGATCGCGATTAGCGGTTGCAGCAGCAGCGGCAAGACCACCCTGGCGAAATTCCTGGCGAACGCGATCCCGGGTTGCATCCTGATTCACGAGGACGATTTTTACAAGCCGGATAGCGAGATCCCGATTAACGAAAAATATGGTGTGGCGGACTGGGATTGCCCGGAGGCGCTGGACCTGGATGCGTTCAAGCGTGAACTGGACCTGATCAAAACCACCGGTAGCATCAAGACCAAACTGATTCACAACGAGAACGTTGACGATATCGGCAAGTTTAACATTAAACAGGAAGACTGGGATGCGCTGCGTGCGAAGCTGAGCAGCGTGATCGAGAGCGACCTGAAAGTGGTTCTGGTTGATGGTTTCATGATTTTTAACGACGAGGAACTGATGCGTAAGTTCGATATCCGTATTTTTGTGCGTGCGCCGTACGAAGTTCTGAGCCGTCGTCGTCACGCGCGTGCGGGTTATAAAACCCTGGAGAGCTTCGTGGTGGACCCGCCGTACTATTTCGATGAATTTGTTTACCGTGCGTATCGTGAGGAACACAAGCACCTGTTCGTGAACGAGGATGTTGAAGGTAGCCTGCGTAGCGACGCGGGCCTGTTTGAACTGATCAACGACGATGAGACCGAAATTACCACCGCGCTGAACACCATCGCGGATTACATTGTTAGCCACCTGGACGCGAACCATCATCATCATCATCATTAA; SEQ ID No.10: MTTTKVKLIAISGCSSSGKTTLAKFLANAIPGCILIHEDDFYKPDSEIPINEKYGVADWDCPEALDLDAFKRELDLIKTTGSIKTKLIHNENVDDIGKFNIKQEDWDALRAKLSSVIESDLKVVLVDGFMIFNDEELMRKFDIRIFVRAPYEVLSRRHARAGYKTLESFVVDPPYYFDEFVYRAYREEHKHLFVNEDVEGSLRSDAGLFELINDDETEITTALNTIADYIVSHLDANHHHHHH; SEQ ID No.11: ATGAAGAAGAACATCTACAAGAAAGAACTGTACAAACTGCAAGTTGAACTGGTGAAATTCCAGAAGTACGTGATCGAAGAGAACGTTGCGGTTTGCCTGGTTCTGGAAGGTCGTGACACCGCGGGTAAAGATGGCACCATCAAACGTTTCACCGAACACCTGAGTCCGCGTGAAGCACGTACCGTTGCGCTTGGTGTTCCGTCTGACAAAGAGAAGAAGAGCTGGTACTTCCAGCGTTACGTTCCACATCTGCCGTCCGCAGGTGAAATCGTGTTCTTCAACCGTTCTTGGTACAACCGTGCAGGTGTTGAGAAAGTTATGGGTTTCTGCACTAAGAAACAGTACAAAGCGTTCATGGAAGAAGTTGGTTCTTTCGAACAGATGCTGACTCACTCTAACATCCGTTTCTTCAAATACTACCTGGACATCACGAAGAAAGAACAGAAAAAACGTCTGGAAGCGCGTAAGACCGATCCGCTGAAACAGTGGAAACTGTCTCCGATCGACGCTAAAGCGCAGAAGATGTGGGATGCGTACTCTAAAGCTCGTGACGACATGTTCAACAAGACCAGCTTCATCTACGCGCCGTGGTACGTTGTTCACACCGACGACAAGAAAGAAGCTCGTATCAACATCATGAAACACTTTCTGTCTCTGAACGACTATCCAGACAAAGACAAAGCGCTGCTGGTTTACGACCACGACGTTATCTGCAAATTCGATCCGGTTTGCTACGAGAAAGAAATGATCGCTCCGCTCGAGCACCACCACCACCACCACTGA; SEQ ID No.12: MKKNIYKKELYKLQVELVKFQKYVIEENVAVCLVLEGRDTAGKDGTIKRFTHEHLSPREARTVALGVPSDKEKKSWYFQRYVPHLPSAGEIVFFNRSWYNRAGVEKVVMGFCTKKQYKAFMEEVGSFEQMLT HSNIRFFKYYLDITKKEQKKRLEARKTDPLKQWKLSPIDAQQKMWDAYSKARDDMFNKTSFIYAPWYVVHTDDKKEARINIMKHFLSLNDYPDKDKALLVYDHDVICKFDPVCYEKEMIAPLEHHHHHH; SEQ ID No.13: ATGACCACCACCAAGGTGAAACTGATCGCGATTAGCGGTTGCAGCAGCAGCGGCAAGACCACCCTGGCGAAATTCCTGGCGAACGCGATCCCGGGTTGCATCCTGATTCACGAGGACGATTTTTACAAGCCGGATAGCGAGATCCCGATTAACGAAAAATATGGTGTGGCGGACTGGGATTGCCCGGAGGCGCTGGACCTGGATGCGTTCAAGCGTGAACTGGACCTGATCAAAACCACCGGTAGCATCAAGACCAAACTGATTCACAACGAGAACGTTGACGATATCGGCAAGTTTAACATTAAACAGGAAGACTGGGATGCGCTGCGTGCGAAGCTGAGCAGCGTGATCGAGAGCGACCTGAAAGTGGTTCTGGTTGATGGTTTCATGATTTTTAACGACGAGGAACTGATGCGTAAGTTCGATATCCGTATTTTTGTGCGTGCGCCGTACGAAGTTCTGAGCCGTCGTCGTCACGCGCGTGCGGGTTATAAAACCCTGGAGAGCTTCGTGGTGGACCCGCCGTACTATTTCGATGAATTTGTTTACCGTGCGTATCGTGAGGAACACAAGCACCTGTTCGTGAACGAGGATGTTGAAGGTAGCCTGCGTAGCGACGCGGGCCTGTTTGAACTGATCAACGACGATGAGACCGAAATTACCACCGCGCTGAACACCATCGCGGATTACATTGTTAGCCACCTGGACGCGAACCATCATCATCATCATCATGAAGCGGCGGCGAAGGGCTTTGGCTGCCCGGAAGATGAATATGAATGCCATAATCATTGCAAAAATAGCGTGGGCTGCCGTGGCGGCTATTGCGATGCAGGCACCCTGCGTCAGCGTTGCACCTGCTATGGCTGCAATCGTAAAGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGACACCACCACCACCACCACTGA; SEQ ID No.14: MTTTKVKLIAISGCSSSGKTTLAKFLANAIPGCILIHEDDFYKPDSEIPINEKYGVADWDCPEALDLDAFKRELDLIKTTGSIKTKLIHNENVDDIGKFNIKQEDWDALRAKLSSVIESDLKVVLVDGFMIFNDEELMRKFDIRIFVRAPYEVLSRRHARAGYKTLESFVVDPPYYFDEFVYRAYREEHKHLFVNEDVEGSLRSDAGLFELINDDETEITTALNTIADYIVSHLDANHHHHHHEAAKGFGCPEDEYECHNHCKNSVGCRGGYCDAGTLRQRCTCYGCNRKELRRQACGRTRHHHHHH; SEQ ID No.15: ATGAAGAAGAACATCTACAAGAAAGAACTGTACAAACTGCAAGTTGAACTGGTGAAATTCCAGAAGTACGTGATCGAAGAGAACGTTGCGGTTTGCCTGGTTCTGGAAGGTCGTGACACCGCGGGTAAAGATGGCACCATCAAACGTTTCACCGAACACCTGAGTCCGCGTGAAGCACGTACCGTTGCGCTTGGTGTTCCGTCTGACAAAGAGAAGAAGAGCTGGTACTTCCAGCGTTACGTTCCACATCTGCCGTCCGCAGGTGAAATCGTGTTCTTCAACCGTTCTTGGTACAACCGTGCAGGTGTTGAGAAAGTTATGGGTTTCTGCACTAAGAAACAGTACAAAGCGTTCATGGAAGAAGTTGGTTCTTTCGAACAGATGCTGACTCACTCTAACATCCGTTTCTTCAAATACTACCTGGACATCACGAAGAAAGAACAGAAAAAACGTCTGGAAGCGCGTAAGACCGATCCGCTGAAACAGTGGAAACTGTCTCCGATCGACGCTAAAGCGCAGAAGATGTGGGATGCGTACTCTAAAGCTCGTGACGACATGTTCAACAAGACCAGCTTCATCTACGCGCCGTGGTACGTTGTTCACACCGACGACAAGAAAGAAGCTCGTATCAACATCATGAAACACTTTCTGTCTCTGAACGACTATCCAGACAAAGACAAAGCGCTGCTGGTTTACGACCACGACGTTATCTGCAAATTCGATCCGGTTTGCTACGAGAAAGAAATGATCGCTCCGGAAGCGGCGGCGAAGGGCTTTGGCTGCCCGGAAGATGAATATGAATGCCATAATCATTGCAAAAATAGCGTGGGCTGCCGTGGCGGCTATTGCGATGCAGGCACCCTGCGTCAGCGTTGCACCTGCTATGGCTGCAATCGTAAAGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGACACCACCACCACCACCACTGA; SEQ ID No.16: MKKNIYKKELYKLQVELVKFQKYVIEENVAVCLVLEGRDTAGKDGTIKRFTEHLSPREARTVALGVPSDKEKKSWYFQRYVPHLPSAGEIVFFNRSWYNRAGVEKVVMGFCTKKQYKAFMEEVGSFEQMLTHSNIRFFKYYLDITKKEQKKRLEARKTDP LKQWKLSPIDAQQKMWDAYSKARDDMFNKTSFIYAPWYVVHTDDKKEARINIMKHFLSLNDYPDKDKALLVYDHDVICKFDPVCYEKEMIAPEAAAKGFGCPEDEYECNHCKNSVGCRGGYCDAGTLRQRCTCYGCNRKELRRQACGRTRHHHHHH; SEQ ID No.17: ATGGTGAGCAAAGGCGAGGAACTGTTCACCGGTGTGGTTCCGATCCTGGTTGAGCTGGACGGCGATGTGAACGGTCACAAGTTTAGCGTTAGCGGCGAGGGCGAAGGTGACGCGACCTACGGCAAGCTGACCCTGAAATTCATTTGCACCACCGGTAAACTGCCGGTTCCGTGGCCGACCCTGGTGACCACCCTGACCTACGGTGTGCAGTGCTTTAGCCGTTATCCGGACCACATGAAGCAACACGATTTCTTTAAAAGCGCGATGCCGGAGGGCTACGTTCAGGAACGTACCATCTTCTTTAAGGACGATGGTAACTATAAAACCCGTGCGGAAGTGAAGTTCGAAGGCGACACCCTGGTGAACCGTATCGAGCTGAAGGGTATTGACTTTAAAGAAGATGGCAACATTCTGGGTCACAAGCTGGAGTACAACTATAACAGCCACAACGTTTATATCATGGCGGATAAGCAGAAAAACGGCATTAAGGTGAACTTTAAAATCCGTCACAACATTGAAGACGGTAGCGTTCAACTGGCGGATCACTACCAGCAAAACACCCCGATTGGTGATGGTCCGGTGCTGCTGCCGGATAACCACTATCTGAGCACCCAGAGCGCGCTGAGCAAGGACCCGAACGAGAAACGTGATCACATGGTTCTGCTGGAATTCGTGACCGCGGCGGGTATCACCCTGGGTATGGACGAACTGTACAAG; SEQ ID No.18: MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK。

[0062] Example 1: Selection of anchored peptide mutants Four anchoring peptide mutants, CgDef(V1)(T36C), CgDef(V2)(R32C), CgDef(V3)(P5V), and CgDef(V4)(T36V), as well as the original anchoring peptide CgDef(WT), were selected and linked to enhanced green fluorescent protein (eGFP) using a one-step cloning technique. Plasmid expression vectors pET-28a(+) / CgDef(V1)-eGFP, pET-28a(+) / CgDef(V2)-eGFP, pET-28a(+) / CgDef(V3)-eGFP, pET-28a(+) / CgDef(V4)-eGFP, and pET-28a(+) / CgDef(WT)-eGFP were constructed and transformed to obtain recombinant *E. coli* BL21(DE3) / pET-28a(+) / CgDef(V1)-eGFP and *E. coli* BL21(DE3)-eGFP. BL21(DE3) / pET-28a(+) / CgDef(V2)-eGFP, E. coli BL21(DE3) / pET-28a(+) / CgDef(V3)-eGFP, E. coli BL21(DE3) / pET-28a(+) / CgDef(V4)-eGFP, and E. coli BL21(DE3) / pET-28a(+) / CgDef(WT)-eGFP were induced to express the corresponding wet bacterial cells.

[0063] Take 0.5g of the above wet bacterial cells and add 10mL of phosphate buffer (50mM, pH 8.0) to dissolve by shaking. Dissolve the strain by sonication (250W, 20min), centrifuge to obtain eGFP-CgDef cell lysate. Take 1mL of each lysate and add it to a 0.5cm*0.5cm sheet of anchoring material (PP, polypropylene). Incubate at 37℃ for 30min to obtain conjugates of eGFP-CgDef(WT), eGFP-CgDef(V1), eGFP-CgDef(V2), eGFP-CgDef(V3), and eGFP-CgDef(V4) with the anchoring material. Wash the prepared conjugates three times with ddH2O and place them in 96-well black microplates without caps. Based on the characteristics of the enhanced green fluorescent protein contained therein, detect them using a TECAN fluorescence detector with wavelengths of λex = 488nm and λem = 525nm. Figure 1As shown, the fluorescence intensities of eGFP-CgDef(V1), eGFP-CgDef(V2), eGFP-CgDef(V3), and eGFP-CgDef(V4) were all higher than that of eGFP-CgDef(WT); among them, eGFP-CgDef(V3) exhibited the strongest fluorescence intensity, indicating that CgDef(V3) has the best ability to immobilize with anchoring materials. Therefore, the mutant CgDef(V3) (P5V) was selected as the target to construct fusion proteins with nicotinamide ribokinase and polyphosphate kinase, respectively.

[0064] Example 2: Construction of recombinant plasmids containing anchor peptide mutant genes 1. The fifth proline in the original anchor peptide amino acid sequence (as shown in SEQ ID No. 2) was mutated to valine to obtain the anchor peptide mutant used in this embodiment (CgDef(V3), abbreviated as CgDef in Example 2 and the following examples, with a nucleotide sequence as shown in SEQ ID No. 3 and an amino acid sequence as shown in SEQ ID No. 4). The primers used are shown in Table 1.

[0065] 2. Using the nucleotide sequence of the nicotinamide ribokinase encoding gene shown in SEQ ID No. 9 as a template, upstream and downstream primers were designed, and the primer sequences are shown in Table 1. The anchoring peptide mutant obtained in step 1 was added to the C-terminus and N-terminus of nicotinamide ribokinase to obtain the gene fragment of the recombinant nicotinamide ribokinase fusion protein after the anchoring peptide mutant and nicotinamide ribokinase were combined. The connection between the anchoring peptide mutant gene and the nicotinamide ribokinase gene was confirmed by gel electrophoresis. Subsequently, the template was digested to construct the recombinant plasmids pET-28a(+) / NRK-CgDef and pET-28a(+) / CgDef-NRK. Using the nucleotide sequence of the nicotinamide ribokinase encoding gene shown in SEQ ID No. 9 as a template, upstream and downstream primers were designed, and the primer sequences are shown in Table 1. The gene fragment of the recombinant polyphosphate kinase fusion protein after the anchoring peptide mutant and polyphosphate kinase were constructed using the same method as above, and the recombinant plasmid pET-28a(+) / PPK-CgDef was constructed.

[0066] 3. Design linker peptides. Select flexible linker peptides with the amino acid sequence GGGGS (hereinafter referred to as S) and rigid linker peptides with the amino acid sequence EAAAK (hereinafter referred to as R). Select linker peptides with multiples of 1, 2, and 3, that is, the linker peptides used are: EAAAK (R1), EAAAKEAAAK (R2), EAAAKEAAAKEAAAK (R3), GGGGS (S1), GGGGSGGGGS (S2), and GGGGSGGGGSGGGGS (S3). The gene encoding the linker peptide was inserted into the recombinant plasmids pET-28a(+) / NRK-CgDef and pET-28a(+) / CgDef-NRK using a one-step cloning method, resulting in the recombinant plasmids pET-28a(+) / NRK-R1-CgDef (abbreviated as KR1D), pET-28a(+) / NRK-R2-CgDef, pET-28a(+) / NRK-R3-CgDef, pET-28a(+) / NRK-S1-CgDef, and pET-28a( The gene encoding the linker peptide EAAAK was inserted into the recombinant plasmid pET-28a(+) / NRK-S2-CgDef, pET-28a(+) / NRK-S3-CgDef, pET-28a(+) / CgDef-R1-NRK, pET-28a(+) / CgDef-R2-NRK, pET-28a(+) / CgDef-R3-NRK, pET-28a(+) / CgDef-S1-NRK, pET-28a(+) / CgDef-S2-NRK, and pET-28a(+) / CgDef-S3-NRK. The gene encoding the linker peptide EAAAK was inserted into the recombinant plasmid pET-28a(+) / PPK-CgDef using a one-step cloning method, resulting in the recombinant plasmid pET-28a(+) / PPK-R1-CgDef (abbreviated as PR1D).

[0067] The one-step cloning PCR reaction system is as follows: 2×PhantaMax buffer: 25μL; dNTPs: 1μL; upstream primer: 2μL; downstream primer: 2μL; template: 1μL; PhantaMax Super... Fidelity DNA polymerase: 0.5 μL; ddH2O: 18.5 μL. PCR reaction conditions: pre-denaturation 95℃, 5 min; denaturation 95℃, 30 s; annealing 58℃, 30 s; extension 72℃, 3 min, for a total of 30 cycles; final extension 72℃, 10 min; stored at 4℃. PCR results were verified by DNA agarose gel electrophoresis.

[0068] Table 1. Anchor peptide insertion sites and primer sequences

[0069] 4. Add 5 μL of each of the 14 recombinant plasmids containing the inserted linker peptide gene to pre-thawed competent E. coli BL21(DE3) cells. Incubate on ice for 30 min, then heat shock at 42°C for 90 s, followed by another 3-5 min on ice. Add 300 μL of LB liquid medium and incubate at 37°C for 1 h. After centrifugation at 12000 rpm for 2 min, remove 200 μL of supernatant and resuspend the cells. Take 50 μL of the culture medium and inoculate it onto LB solid medium containing 0.5 μg / mL kanamycin, spreading it evenly. Incubate at 37°C for 12-14 h. Bacterial selection and sequencing verification yielded recombinant *E. coli* strains: *E. coli* BL21(DE3) / pET28a(+) / NRK-Rn-CgDef, *E. coli* BL21(DE3) / pET-28a(+) / NRK-Sn-CgDef, *E. coli* BL21(DE3) / pET-28a(+) / CgDef-Rn-NRK, *E. coli* BL21(DE3) / pET-28a(+) / CgDef-Sn-NRK, where n is 1, 2, or 3, as well as recombinant *E. coli* BL21(DE3) / pET28a(+) / PPK-R1-CgDef. Under the same conditions, the original strain *E. coli* BL21(DE3) / pET-28a(+) / NRK was constructed. SDS-PAGE electrophoresis images of whole cells and cell lysate supernatants of 12 recombinant Escherichia coli strains and the original strain are shown below. Figure 2 , 3 As shown, the relative molecular masses of NRK, DS1K and KS1D, DS2K and KS2D, DS3K and KS3D, DR1K and KR1D, DR2K and KR2D, and DR3K and KR3D are 28.02, 33.11, 33.43, 33.74, 33.27, 33.74, and 34.21 (kDa), respectively, consistent with the electrophoresis results. Furthermore, the expression level of the fusion protein is lower than that of the wild-type enzyme protein, indicating that the introduction of the anchoring peptide reduces the expression level of this enzyme protein in *E. coli*.

[0070] Example 3: Determination of relative enzyme activity of twelve fusion proteins The recombinant Escherichia coli strains E. coli BL21(DE3) / pET-28a(+) / NRK-Rn-CgDef, E. coli BL21(DE3) / pET-28a(+) / NRK-Sn-CgDef, E. coli BL21(DE3) / pET-28a(+) / CgDef-Rn-NRK, E. coli BL21(DE3) / pET-28a(+) / CgDef-Sn-NRK (where n is 1, 2, or 3), and E. coli BL21(DE3) / pET-28a(+) / NRK strain constructed in Example 1 and stored at -80℃ were taken out and inoculated onto LB agar plates containing 0.5 μg / mL kanamycin resistance. After incubation at 37℃ for 12-14 h, single colonies were obtained. Single colonies were picked and placed into 10 mL of LB medium containing 0.5 μg / mL kanamycin. After incubation at 37°C for 8 h, 2 mL of the culture was transferred to 100 mL of fermentation medium containing 0.5 μg / mL kanamycin. After incubation at 37°C for 2 h, 100 μL of IPTG (final concentration 0.1 mM) was added, and enzyme production was induced at 28°C for 12–14 h. The cells were collected by centrifugation at 8000 rpm for 10 min and resuspended in phosphate buffer (50 mM, pH 8.0) to a cell concentration of 50 g / L. The cells were sonicated at 250 W until the solution was clear, centrifuged at 12000 rpm for 30 min, and the supernatant was discarded to obtain 12 crude enzyme solutions. The relative enzyme activity was determined using E. coli BL21(DE3) / pET-28a(+) / NRK as the standard. A crude enzyme solution with a final concentration of 2 g / L was added to a 1 mL reaction system (200 mM nicotinamide ribose (NR), 300 mM adenosine-5-triphosphate disodium salt (ATP), 50 mM magnesium chloride). The reaction was carried out at 40℃ and 1000 rpm for 3 min. 10 μL of the reaction solution was then removed, and 10 μL of H3PO4 (0.2 M) was added to terminate the reaction. The activity was detected by HPLC, and the results were analyzed according to the standard curve (e.g., ...). Figure 5 (As shown) The enzyme activity was calculated by analyzing the NMN content in the sample for preliminary screening.

[0071] Table 2. Relative enzyme activities of recombinant strains containing different linker peptides

[0072] As shown in Table 2, the relative enzyme activity results indicate that KR1D has the highest enzyme activity, which is 54.81% of the wild-type enzyme of the original strain. The enzyme activities of KR2D, KR3D, DR1K, and KS1D are 52.19%, 46.53%, 51.74%, and 45.41% of the wild-type enzyme of the original strain, respectively. These five fusion proteins have relatively high enzyme activities and are used to screen for anchoring effects. The enzyme activities of other fusion proteins are less than 40% of the wild-type enzyme of the original strain.

[0073] Example 4: Comparison of anchoring effects of five fusion proteins with high free enzyme activity Immobilized enzymes were prepared by combining five fusion proteins with PP hollow fiber membrane material. The anchoring effect was compared by detecting the relative enzyme activity changes of six batches of the immobilized enzymes. 1 mL of crude enzyme solution of the five free enzymes with high activity (KR1D, KR2D, KR3D, DR1K, and KS1D) obtained in Example 3 was used for later use.

[0074] Take 0.01g of polypropylene hollow fiber membrane material (PP for short, specifications: inner / outer diameter: 0.4 / 0.6mm, molecular weight cutoff: 100000Da), sonicate with acetone and ethanol for 20 minutes each, wash with water and dry for later use.

[0075] 1 mL of crude enzyme solution was incubated with the anchoring material at 26℃ and 500 rpm for 2 h. Solid-liquid separation was performed, and the resulting enzyme-material conjugates were washed three times with buffer to obtain five immobilized enzymes (KR1D-PP, KR2D-PP, KR3D-PP, DR1K-PP, and KS1D-PP) on the same anchoring material. The five immobilized enzymes were added to 1 mL of phosphate buffer (pH 8.0). A reaction system was constructed using 200 mM nicotinamide riboside (NR) as the substrate, 300 mM adenosine-5-triphosphate disodium salt (ATP) as the phosphate donor, and 50 mM magnesium chloride as the activator. The reaction was carried out at 40℃ and 1000 rpm for 10 min. 10 μL of the reaction solution was added to 10 μL of H3PO4 (0.2 M) to terminate the reaction. HPLC analysis was performed, and the results were analyzed according to the standard curve (e.g., ...). Figure 5 (As shown) The NMN content in the sample was analyzed. Each time the reaction solution was removed, new reaction solution was added, and the reaction was repeated six times. Using the result of the first reaction as the standard, a relative enzyme activity change graph was generated for six batches to determine the batch stability of immobilized enzymes made from different fusion proteins. Figure 6 As shown, KR1D exhibits the best relative enzyme activity stability, retaining 83.24% of its initial activity after 6 batches. KS1D also shows relatively good enzyme activity stability, retaining 65.62% of its initial activity after 6 batches, while the other three retain less than 35% of their initial activity after 6 batches.

[0076] Example 5: Binding of recombinant nicotinamide ribokinase fusion protein (KR1D) based on anchoring peptide mutants to different anchoring materials. The crude KR1D enzyme solution obtained in Example 3 was used to detect protein concentration and content using a BCA assay kit. 1 mL of the crude enzyme solution was taken for later use. BCA assay kit protein detection method: The concentration of protein in the supernatant was tested using a BCA protein assay kit for colorimetric reaction, and then the absorbance of the sample at 562 nm was measured using a microplate reader (absorbance is directly proportional to the standard protein content). Finally, an instantaneous standard curve was plotted. The absorbance of the crude enzyme solution at 562 nm was tested, and the results were compared with the standard curve (…). Figure 4 (The protein content in the crude enzyme solution is calculated by re-preparing the solution for each measurement.)

[0077] The following materials were treated using the same method as that used for polypropylene (PP) in Example 4: 0.01g of polyvinylidene fluoride hollow fiber membrane material (PVDF, specifications: inner / outer diameter: 0.9 / 1.5mm, molecular weight cutoff: 6000Da), 0.01g of polysulfone hollow fiber membrane material (PSF, specifications: inner / outer diameter: 0.9 / 1.4mm, molecular weight cutoff: 10000Da), and 0.01g of polytetrafluoroethylene hollow fiber membrane material (PTFE, specifications: inner / outer diameter: 0.3 / 0.8mm).

[0078] 1 mL of crude enzyme solution was incubated with different anchoring materials at 26 °C and 500 rpm for 2 h. After solid-liquid separation, the concentration and content of the remaining protein were detected using a BCA kit. The enzyme-material conjugates were washed three times with buffer to obtain immobilized enzymes (KR1D-PP, KR1D-PVDF, KR1D-PSF, KR1D-PTFE), which were stored at 4 °C for later use.

[0079] The enzyme protein content in the supernatant before and after immobilization was determined, and the protein adsorption rate was calculated. The calculation formula is as follows: Protein adsorption rate =

[0080] Following the above method, the protein adsorption rate of KR1D fusion protein with different anchoring materials was measured. The results are shown in Table 3. It can be seen that PP hollow fiber membrane filaments have the best effect as anchoring material, with a protein adsorption rate as high as 89.0%. PVDF, PSF, and PTFE materials also showed good anchoring effects: the protein adsorption rate of recombinant nicotinamide ribokinase fusion protein anchored with PVDF was 61.2%; the protein adsorption rate of recombinant nicotinamide ribokinase fusion protein anchored with PSF was 54.5%; and the protein adsorption rate of recombinant nicotinamide ribokinase fusion protein anchored with PTFE was 48.4%.

[0081] Table 3 Adsorption rates (%) of different fusion proteins with different anchoring materials

[0082] Example 6: Comparison of batch reaction enzyme activity and stability of KR1D fusion protein conjugates with different polymer materials as immobilized enzymes. Following the method described in Example 5, four immobilized enzymes (KR1D-PP, KR1D-PVDF, KR1D-PSF, and KR1D-PTFE) were obtained by binding four different anchoring materials to KR1D. Each of the four immobilized enzymes was added to 1 mL of phosphate buffer (pH 8.0). The reaction system consisted of 200 mM nicotinamide ribose (NR), 300 mM adenosine-5-triphosphate disodium salt (ATP), and 50 mM magnesium chloride. The reaction was carried out at 40°C and 1000 rpm for 10 min. 10 μL of the reaction solution was taken and 10 μL of H3PO4 (0.2 M) was added to terminate the reaction. The results were analyzed by HPLC according to a standard curve (e.g., Figure 5 (As shown) The NMN content in the sample was analyzed, and the enzyme activity and specific activity were calculated. Each time the reaction solution was removed, fresh reaction solution was added, and the reaction was repeated six times to create six batches of enzyme specific activity charts, in order to determine the enzyme activity stability of immobilized enzymes made from different fusion proteins. Figure 7 As shown, KR1D-PP exhibited the highest specific enzyme activity, with an average specific enzyme activity of 1897.03 U in the first batch of reactions. The specific enzyme activity in the sixth batch decreased by 16.76% compared to the first batch. The average specific enzyme activities of KR1D-PVDF, KR1D-PSF, and KR1D-PTFE in the first batch of reactions were 1143.90 U, 946.19 U, and 733.87 U, respectively. The specific enzyme activities in the sixth batch decreased by 54.0%, 53.3%, and 49.8% compared to the first batch, respectively.

[0083] Example 7: Production of NMN by immobilized enzyme (KR1D-PP) at a substrate concentration of 20 mM and batch reaction stability Using NR as a substrate, NMN was produced by catalysis with an immobilized enzyme (KR1D-PP). The specific procedure was as follows: KR1D-PP prepared as in Example 5 was added to 1 mL of phosphate buffer (pH 8.0). The reaction system consisted of 20 mM nicotinamide riboside (NR) as the substrate, 30 mM adenosine-5-triphosphate disodium salt (ATP), and 5 mM magnesium chloride. The reaction was carried out at 40°C and 1000 rpm for 2 h. After the reaction, 10 μL of the reaction solution was taken and 10 μL of H3PO4 (0.2 M) was added to terminate the reaction. The results were detected by HPLC and analyzed according to a standard curve (e.g., ...). Figure 5 (As shown) The content of NMN in the sample was analyzed.

[0084] After 2 hours of reaction, the reaction solution was aspirated and separated, and then added back into a reaction system consisting of 20 mM NR, 30 mM ATP, and 5 mM magnesium chloride as substrates, repeating the first reaction process. After multiple repetitions, the NMN production amount for each reaction was calculated and compared. The NMN yield at 2 hours after 40 repetitions of KR1D-PP is shown below. Figure 8 As shown, under reaction conditions with a 20 mM nicotinamide riboside substrate concentration, the NMN yield for the first 10 batches after 2 hours was greater than or equal to 81%, the yield for the 11th batch was 76%, the yield dropped to 70% after the 15th batch, and the yield was less than 50% after the 30th batch.

[0085] Example 8: Production of NMN from immobilized enzyme (KR1D-PP) at a substrate concentration of 100 mM and batch reaction stability Using NR as a substrate, NMN was produced by catalysis with an immobilized enzyme (KR1D-PP). The specific procedure was as follows: KR1D-PP prepared as in Example 5 was added to 1 mL of phosphate buffer (pH 8.0). The reaction system consisted of 100 mM nicotinamide riboside (NR) as the substrate, 150 mM adenosine-5-triphosphate disodium salt (ATP), and 25 mM magnesium chloride. The reaction was carried out at 40°C and 1000 rpm for 6 h. After the reaction, 10 μL of the reaction solution was taken and 10 μL of H3PO4 (0.2 M) was added to terminate the reaction. The results were detected by HPLC and analyzed according to a standard curve (e.g., ...). Figure 5 (As shown) The content of NMN in the sample was analyzed.

[0086] After 6 hours of reaction, the reaction solution was aspirated and separated, and then added back into a reaction system consisting of 100 mM NR, 150 mM ATP, and 25 mM magnesium chloride as substrates, repeating the first reaction process. After multiple repetitions, the NMN production amount for each reaction was calculated and compared. The NMN yield at 6 hours after 40 repetitions of KR1D-PP is shown below. Figure 9 As shown, under reaction conditions with a nicotinamide riboside substrate concentration of 100 mM, the yields for the first 9 batches over 6 hours were greater than or equal to 86%, the yield for the 10th batch was 80%, the yield dropped to 74% after the 15th batch, and the yield was less than 50% after the 30th batch.

[0087] Example 9: Comparison of thermostability and storage stability between immobilized enzyme (KR1D-PP) and free enzyme (KR1D) 1. Take the crude KR1D enzyme solution obtained in Example 3 and the KR1D-PP immobilized enzyme obtained in Example 6, incubate them at a temperature of 20-60℃ for 2 hours, and then measure the residual enzyme activity. The thermal stability is observed with the enzyme activity of the untreated free enzyme and the immobilized enzyme as 100%.

[0088] 2. The crude KR1D enzyme solution obtained in Example 3 and the immobilized KR1D enzyme obtained in Example 6 were stored at 4°C. The residual enzyme activity was measured every 5 days, with the initial enzyme activity of both being 100%, to assess the difference in stability during low-temperature storage. The method for determining the free enzyme activity is described in Example 3, and the method for determining the immobilized enzyme activity is described in Example 6.

[0089] After incubating at a gradient temperature of 20-60℃ for 2 hours each, as follows Figure 10 As shown, the enzyme activities of immobilized and free enzymes decreased to varying degrees, with the immobilized enzyme showing a smaller decrease and the free enzyme showing a larger and more pronounced decrease, demonstrating that the immobilized enzyme has better thermostability than the free enzyme. The enzyme activities of immobilized and free enzymes were measured after storage at 4°C for different numbers of days to demonstrate their storage stability. Figure 11 As shown, after the same number of storage days, the residual enzyme activity of the immobilized enzyme was higher than that of the free enzyme, demonstrating the better storage stability of the immobilized enzyme.

[0090] Example 10: Production of NMN from immobilized enzyme (KR1D-PP-PR1D) at a substrate concentration of 100 mM and batch reaction stability Take 0.5g of each of the wet bacterial cells *E. coli* BL21(DE3) / pET-28a(+) / NRK-R1-CgDef and *E. coli* BL21(DE3) / pET-28a(+) / PPK-R1-CgDef obtained in Example 2, dissolve them in 10mL of buffer (50mM, pH 8.0, K2HPO4-KH2PO4), shake thoroughly to dissolve, and sonicate under ice bath conditions. The sonication cell disruptor was set to 250W, operating for 1 second followed by a 2-second interval, until the solution became clear. Subsequently, the cell disruption solution was centrifuged at 12000 rpm at 4℃ for 30 min to remove cell debris precipitate. The supernatant was collected as crude enzyme solution, and the protein content was determined using a BCA assay kit. 1mL of the crude enzyme solution was reserved for later use. The crude enzyme solution was incubated with 0.01g of treated PP at 26℃ and 500rpm for 2h. After solid-liquid separation, the remaining protein content was detected using a BCA kit. The enzyme-material conjugate was washed three times with buffer to obtain immobilized enzyme (KR1D-PP-PR1D), which was stored at 4℃ for later use.

[0091] NMN was produced using NR as a substrate and an immobilized enzyme (KR1D-PP-PR1D) as a reaction medium. The specific procedure was as follows: KR1D-PP-PR1D was added to 1 mL of phosphate buffer (pH 8.0). The reaction system consisted of 100 mM nicotinamide riboside (NR) as the substrate, 15 mM adenosine-5-triphosphate disodium salt (ATP), and 25 mM magnesium chloride. The reaction was carried out at 40℃ and 1000 rpm for 6 h. After the reaction, 10 μL of the reaction solution was taken and 10 μL of H3PO4 (0.2 M) was added to terminate the reaction. The results were detected by HPLC and analyzed according to a standard curve (e.g., ...). Figure 5 (As shown) The content of NMN in the sample was analyzed.

[0092] After 6 hours of reaction, the reaction solution was aspirated and separated, and then added back into a reaction system consisting of 100 mM NR, 15 mM ATP, and 25 mM magnesium chloride as substrates, repeating the first reaction process. After multiple repetitions, the NMN production amount for each reaction was calculated and compared. The NMN yield of KR1D-PP-PR1D after 40 repetitions of 6 hours of reaction is as follows. Figure 12 As shown, the yield in 6 hours was greater than or equal to 82% for the first 9 batches, 81% for the 10th batch, dropped to 68% after the 15th batch, and was less than 51% after the 30th batch.

[0093] Example 11: Design of a continuous flow biocatalytic membrane reactor for NMN production using immobilized enzyme (KR1D-PP-PR1D) 7.5g of the wet bacterial cells *E. coli* BL21(DE3) / pET-28a(+) / NRK-R1-CgDef and *E. coli* BL21(DE3) / pET-28a(+) / PPK-R1-CgDef obtained in Example 2 were dissolved in 150mL of phosphate buffer (50mM, pH 8.0, K2HPO4-KH2PO4). The solution was thoroughly shaken and dissolved. The cells were then sonicated under ice bath conditions. The sonication cell disruptor was set to 250W, operating for 1 second followed by a 2-second interval until the solution was clear. The cell disruption solution was then centrifuged at 12000 rpm at 4℃ for 30 min to remove cell debris. The supernatant was collected as crude enzyme solution, and the protein content was determined using a BCA assay kit. 150mL of the crude enzyme solution was reserved. A PP hollow fiber membrane reactor (specifications: Φ63*300mm, membrane area: 0.75m²) was used. 2 The crude enzyme solution was introduced into the reactor using a peristaltic pump and silicone tubing, and incubated at room temperature for 2 hours. Then, it was rinsed three times with buffer solution using a peristaltic pump and set aside for later use.

[0094] Using NR as a substrate, NMN is continuously produced in the reactor described above. Figure 13The specific operation is as follows: Prepare a reaction system (300 mL) using 200 mM nicotinamide ribose (NR) as the substrate, 30 mM adenosine-5-triphosphate disodium salt (ATP), and 50 mM magnesium chloride. Introduce the system into the reactor at 40°C with a peristaltic pump running rapidly. Every 10 min, take 10 μL of the reaction solution and add 10 μL of H3PO4 (0.2 M) to terminate the reaction. Detect the reaction by HPLC, according to the standard curve (e.g., ...). Figure 5 The NMN content in the sample (as shown) was analyzed. The space-time yield of this biocatalytic membrane reactor was calculated to be 1.604 × 10⁻⁶. 3 g·L -1 ·d -1 .

[0095] Comparative Example 1: Binding of recombinant nicotinamide ribokinase fusion protein based on original anchoring peptide to different anchoring materials Recombinant *E. coli* strains containing the original anchoring peptide encoding gene were constructed using the method described in Example 2, yielding *E. coli* BL21(DE3) / pET-28a(+) / NRK-R1-CgDef(WT) and *E. coli* BL21(DE3) / pET-28a(+) / PPK-R1-CgDef(WT). Following the same anchoring materials and experimental methods as in Example 5, the protein adsorption rate of the fusion proteins based on the original anchoring peptide with different anchoring materials was determined, and two immobilized enzymes (KR1D-WT-PP and KR1D-WT-PP-PR1D-WT) were prepared. The results are shown in Table 4. Compared with the recombinant nicotinamide ribokinase fusion protein based on the anchoring peptide mutant, the protein adsorption rate of the recombinant nicotinamide ribokinase fusion proteins based on the original anchoring peptide decreased to varying degrees when bound to different anchoring materials. The protein adsorption rate of the PP-anchored conjugate decreased to 71.1%, that of PVDF decreased to 50.2%, that of PSF decreased to 42.6%, and that of PTFE decreased to 39.4%.

[0096] Table 4 Adsorption rates (%) of different fusion proteins with different anchoring materials

[0097] Comparative Example 2: Production of NMN by the original anchored peptide-mediated immobilized enzyme (KR1D-WT-PP) at a substrate concentration of 100 mM. Using NR as a substrate and the immobilized enzyme (KR1D-WT-PP) based on the original anchored peptide prepared in Comparative Example 1 as a biocatalyst, the reaction was carried out as follows: KR1D-WT-PP was added to 1 mL of phosphate buffer (pH 8.0). The reaction system consisted of 100 mM nicotinamide ribose (NR) as substrate, 150 mM adenosine-5-triphosphate disodium salt (ATP), and 25 mM magnesium chloride. The reaction was carried out at 40 °C and 1000 rpm for 6 h. After the reaction, 10 μL of sample was taken and 10 μL of H3PO4 (0.2 M) was added to terminate the reaction. The results were detected by HPLC according to the standard curve (e.g., Figure 5 (As shown) The content of NMN in the sample was analyzed.

[0098] After 6 hours of reaction, the reaction solution was aspirated and separated, and then added back into a reaction system consisting of 100 mM NR, 150 mM ATP, and 25 mM magnesium chloride as substrates, repeating the first reaction process. After multiple repetitions, the NMN production amount for each reaction was calculated and compared. The NMN yield at 6 hours after 40 repetitions of KR1D-WT-PP is as follows. Figure 14 As shown, the yield in 2 hours was greater than or equal to 75% for the first 6 batches, 55% for the 7th batch, 50% for the 8th batch and onwards, and less than 20% for the 30th batch and onwards.

[0099] Comparative Example 3: Production of NMN by the original anchored peptide-mediated immobilized enzyme (KR1D-WT-PP-PR1D-WT) at a substrate concentration of 100 mM. Using NR as a substrate and the immobilized enzyme KR1D-WT-PP-PR1D-WT based on the original anchored peptide prepared in Comparative Example 1 as a biocatalyst, the reaction was carried out as follows: KR1D-WT-PP-PR1D-WT was added to 1 mL of phosphate buffer (pH 8.0). The reaction system consisted of 100 mM nicotinamide ribose (NR) as substrate, 15 mM adenosine-5-triphosphate disodium salt (ATP), and 25 mM magnesium chloride. The reaction was carried out at 40 °C and 1000 rpm for 6 h. After the reaction, 10 μL of the reaction solution was taken and 10 μL of H3PO4 (0.2 M) was added to terminate the reaction. The results were detected by HPLC according to the standard curve (e.g., Figure 5 (As shown) The content of NMN in the sample was analyzed.

[0100] After 6 hours of reaction, the reaction solution was aspirated and separated, and then added back into a reaction system consisting of 100 mM NR, 15 mM ATP, and 25 mM magnesium chloride as substrates, repeating the first reaction process. After multiple repetitions, the NMN production amount for each reaction was calculated and compared. The NMN yield of KR1D-WT-PP-PR1D-WT after 40 repetitions of 6 hours of reaction is shown below. Figure 15As shown, the yield of the first 5 batches at 6 hours was greater than or equal to 73%, the yield of the 9th batch was 50%, the yield dropped to 35% after the 15th batch, and the yield was less than 18% after the 30th batch.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises a functional enzyme protein and an anchoring peptide mutant; the anchoring peptide mutant is attached to the N-terminus or C-terminus of the functional enzyme protein. The anchoring peptide mutant is obtained by mutating proline at position 5 of the amino acid sequence shown in SEQ ID No. 2 to valine; The functional enzyme protein is nicotinamide ribokinase or polyphosphate kinase.

2. The fusion protein according to claim 1, characterized in that, The anchoring peptide mutant is linked to the functional enzyme protein via a linker peptide. The linker peptide is an N-segment repeating sequence of a flexible linker peptide or a rigid linker peptide, where N = 1 to 3; the amino acid sequence of the flexible linker peptide is shown in SEQ ID No. 6; the amino acid sequence of the rigid linker peptide is shown in SEQ ID No.

8.

3. The fusion protein according to claim 2, characterized in that, The linker peptide is one of the following: one rigid linker peptide repeat sequence, two rigid linker peptide repeat sequences, three rigid linker peptide repeat sequences, or one flexible linker peptide repeat sequence.

4. The fusion protein according to any one of claims 1 to 3, characterized in that, The amino acid sequence of the nicotinamide ribokinase is shown in SEQ ID No. 10; the amino acid sequence of the polyphosphate kinase is shown in SEQ ID No.

12. The fusion protein is selected from any of the following: A1) A fusion protein containing the nicotinamide ribokinase, with the amino acid sequence shown in SEQ ID No. 14; A2) A fusion protein containing the polyphosphate kinase described in SEQ ID No. 16, with an amino acid sequence as shown.

5. A gene encoding a fusion protein as described in any one of claims 1 to 4, characterized in that, The nucleotide sequence of the fusion protein encoding gene is selected from any of the following: B1) When the amino acid sequence of the fusion protein is SEQ ID No. 14, its encoding gene is the nucleotide sequence shown in SEQ ID No. 13; B2) When the amino acid sequence of the fusion protein is SEQ ID No. 16, its encoding gene is the nucleotide sequence shown in SEQ ID No.

15.

6. A recombinant expression vector or genetically engineered bacterium comprising the encoding gene as described in claim 5.

7. An immobilized enzyme, characterized in that, include: Polypropylene material; as well as The fusion protein immobilized on the polypropylene material as described in any one of claims 1 to 4; The polypropylene material is preferably in the form of hollow fiber membrane filaments.

8. A method for preparing the immobilized enzyme as described in claim 7, characterized in that, Includes the following steps: S1: Take the crude enzyme solution or purified enzyme solution of the fusion protein; S2: Take polypropylene hollow fiber membrane material, wash and dry it; S3: Mix the crude enzyme solution in S1 with the polypropylene hollow fiber membrane material in S2, and incubate them together to obtain the immobilized enzyme.

9. The immobilized enzyme of claim 7, or the immobilized enzyme prepared by the method of claim 8, in the catalytic production of nicotinamide mononucleotide.

10. The application according to claim 9, characterized in that, The application is carried out in a reaction system containing nicotinamide ribose, ATP, and magnesium ions.

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