A fusion uric acid oxidase and its use in a biosensing element

Abstract

The application provides a fusion uric acid oxidase and application thereof in a biosensing element. The fusion uric acid oxidase comprises a carbohydrate binding module and a uric acid oxidase, and the fusion uric acid oxidase can be made into a biosensing element for uric acid detection, and has the advantages of high sensitivity and good stability.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, specifically relating to a fusion protein constructed by linking a urate oxidase gene with a carbohydrate binding domain gene and its application in biosensing elements. Background Technology

[0002] Uric acid (UA) is an important nitrogenous compound, widely present in biological fluids such as serum and urine, and is the main end product of purine metabolism. Normal serum uric acid levels in healthy individuals are approximately 0.21-0.43 mM in adult men and 0.15-0.36 mM in adult women, while uric acid concentrations in patients with gout can reach 1.49-4.46 mM. Abnormal uric acid levels in body fluids can predict diseases such as gout, metabolic syndrome, and kidney disease; therefore, uric acid is an effective biomarker for detecting purine metabolism disorders. Establishing rapid and reliable methods for detecting uric acid concentrations in body fluids is of great significance for the diagnosis and treatment of diseases.

[0003] Currently, various methods for detecting uric acid (UA) have been developed, such as high-performance liquid chromatography (HPLC), spectrophotometry, ion chromatography, HPLC / isotope dilution mass spectrometry (ID-MS), and electrochemical biosensing. Among these methods, amperometric enzyme biosensors based on the redox catalysis of uricase or uricase (UOX, EC 1.7.3.3) have attracted wider attention due to their advantages of simplicity, sensitivity, specificity, and rapid response. In the biochemical process, UOX immobilized on the electrode consumes O2 and reacts with UA to generate H2O2, allantoin, and CO2. The reaction equation is as follows: The concentration of reduced O2 or oxidized H2O2 is proportional to the concentration of UA. The oxidation or reduction signal is then converted and amplified into a readable physical signal by a transducer and electronic amplifier. Therefore, the high stability and maintained catalytic activity of immobilized UOX are key guarantees for accurate UA sensing.

[0004] In previous studies, different types of enzyme immobilization techniques have been used to construct current-based UA biosensors. Physical adsorption and encapsulation are commonly used methods for immobilizing UOX, but the binding force of adsorption is relatively weak and easily affected by environmental changes, reducing the operational and storage stability of the biosensor. UOX encapsulation is usually achieved using polymers such as polyaniline (PANi) and polypyrrole, but leakage of immobilized enzymes from the polymer poses a challenge to its stability. Methods using bifunctional reagents such as glutaraldehyde or carbodiimide to form stable covalent bonds and cross-links between the enzyme and the carrier or bovine serum albumin (BSA) are also commonly used methods for chemically immobilizing UOX, significantly avoiding enzyme desorption and leakage. However, the widespread enzyme inactivation and low reproducibility caused by covalent bonds are the main limitations of these methods for application in biosensors. In recent years, affinity adsorption has proven to be a promising enzyme immobilization strategy that can improve the performance of enzyme biosensors.

[0005] Carbohydrate-binding domains (CBMs) are continuous amino acid sequences within carbohydrate-active enzymes, representing a class of non-catalytic domains with independently folded structures. CBMs are linked to catalytic domains via linkers, playing a crucial role in substrate recognition and targeted binding within enzyme molecules, exhibiting binding specificity for different types of polysaccharides, particularly cellulose. With advancements in molecular biology, synthetic biology, and computer-aided simulation technologies, researchers are currently utilizing DNA fusion technology to attempt to obtain diverse CBM-enzyme fusion proteins, thereby enhancing enzyme-substrate affinity, enzyme stability, or enzyme activity.

[0006] The applicant has successfully constructed biosensor elements based on the specific binding of CBM and cellulose in previous work, such as patents CN113061189B and CN112851821B. These patents utilize genetic engineering methods to fuse a carbohydrate-binding module (CBM2) from family 2 with glucose oxidase (GOD) and glucose dehydrogenase (GDH), thereby improving glucose sensing performance. However, the construction of biosensor elements for detecting uric acid concentration has not been reported domestically or internationally. Furthermore, the structure and catalytic mechanism of UOX are completely different from those of GOD; therefore, constructing a stable and active CBM-fused UOX as a UA biosensor element remains crucial.

[0007] UOX is a functional tetrameric barrel structure whose active site consists of interfacial residues of two monomers. Each monomer contains two identical antiparallel supersecondary domains with ββααββ, called Tunnelling-fold domains. Certain weak interactions (non-covalent bonds), including van der Waals forces, hydrogen bonds, salt bonds (dissociation bonds), and hydrophobic interactions, participate in stabilizing the quaternary structure. At the tightly packed interfaces between subunits, the complementary arrangement of polar and hydrophobic groups promotes the binding of specific subunits. In the catalytic process of UOX, a coenzyme-free approach has been proposed to promote and control O2 chemistry, suggesting a different catalytic mechanism similar to flavin-dependent oxidases or flavin-dependent monooxygenases, but its mechanism of action is not fully understood. Due to the multi-subunit-dependent catalytic activity and unclear catalytic mechanism of UOX, research on modified UOX has been limited in recent decades. This presents a significant challenge to obtaining stable gene fusion UOX. Summary of the Invention

[0008] To address the aforementioned problems, this invention aims to provide a fusion protein and its application in biosensing elements.

[0009] On one hand, this application provides a fusion protein comprising a carbohydrate-binding module (CBM) and uricase oxidase (UOX).

[0010] In one embodiment, the CBM is selected from CBM2 or CBM3; CBM2 is derived from Thermobifidafusca, whose gene sequence is shown in SEQ ID No. 1 and whose amino acid sequence is shown in SEQ ID No. 2; CBM3 is derived from Acetivibrio thermocellus, whose gene sequence is shown in SEQ ID No. 4 and whose amino acid sequence is shown in SEQ ID No. 5.

[0011] Preferably, the CBM3 is an optimized CBM3, which, relative to the parental CBM3, lacks amino acids 1-4 and 157-159; the amino acid sequence of the parental CBM3 is shown in SEQ ID No. 5; the gene sequence of the optimized CBM3 is shown in SEQ ID No. 5, and the amino acid sequence is shown in SEQ ID No. 6.

[0012] In one embodiment, the UOX is derived from Arthrobacter globiformis, whose gene sequence is shown in SEQ ID No. 7 and whose amino acid sequence is shown in SEQ ID No. 8;

[0013] Preferably, the UOX is an optimized UOX, which, relative to the parental UOX, lacks amino acids 1-3; the amino acid sequence of the parental UOX is shown in SEQ ID No. 8; the gene sequence of the optimized UOX is shown in SEQ ID No. 9, and the amino acid sequence is shown in SEQ ID No. 10.

[0014] In one embodiment, the gene encoding CBM2 is derived from the Thermobifida fusca gene sequence, the gene encoding CBM3 is derived from the Acetivibrio thermocellus gene sequence, and the gene encoding UOX is derived from the Arthrobacter globiformis genome.

[0015] In one embodiment, the CBM is placed at the N-terminal or C-terminal of the UOX.

[0016] Preferably, the CBM is located at the N end of the UOX.

[0017] In one embodiment, the amino acid sequence of the fusion protein is as shown in any of SEQ ID NO.11-15.

[0018] On the other hand, this application also provides a biomaterial, which includes any one or more of the following:

[0019] I. A nucleic acid molecule, wherein the nucleic acid molecule encodes the fusion protein;

[0020] II. An expression cassette, wherein the expression cassette contains the nucleic acid molecule described in I;

[0021] III. A recombinant vector, wherein the recombinant vector contains the nucleic acid molecule described in I or the expression cassette described in II;

[0022] IV. Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in I, the expression cassette described in II, or the recombinant vector described in III.

[0023] In one embodiment, the expression cassette may further include functional elements such as promoters, terminators, and marker genes, as long as they can realize the expression of nucleic acid molecules.

[0024] In one embodiment, the vector is a vector capable of transporting a target gene or exogenous gene into a host cell for amplification and expression, including but not limited to plasmids, bacteriophages or viral vectors, preferably plasmids, such as pET30A.

[0025] In one embodiment, the microorganism may be a host microorganism commonly used in the field of genetic engineering, such as yeast, bacteria, fungi, algae, preferably bacteria, and more preferably Escherichia coli.

[0026] On the other hand, this application also provides the application of the fusion protein in the preparation of biosensing elements or detection kits.

[0027] In one embodiment, the biosensor element is used to detect uric acid concentration.

[0028] In one embodiment, the test kit is a uric acid test kit.

[0029] On the other hand, this application also provides a method for preparing the fusion protein, the method comprising the following steps:

[0030] (a) Transforming the nucleic acid molecule encoding the fusion protein into microorganisms to obtain recombinant engineered bacteria;

[0031] (b) Cultivate the recombinant engineered bacteria to express the fusion protein.

[0032] In one embodiment, the microorganism is Escherichia coli.

[0033] In one embodiment, the cultivation method includes fermentation cultivation.

[0034] In one embodiment, the method further includes a step of purifying the enzyme solution expressed by the recombinant engineered bacteria.

[0035] On the other hand, this application also provides a biosensing element containing the aforementioned fusion protein.

[0036] In one embodiment, the biosensor element further comprises uricase (UOX). Preferably, the UOX is wild-type UOX; more preferably, the amino acid sequence of the UOX is as shown in SEQ ID NO. 8.

[0037] In one embodiment, the biosensing element contains the fusion protein and uricase (UOX).

[0038] On the other hand, this application also provides a method for preparing the biosensing element, the method comprising immobilizing the fusion protein onto the enzyme membrane surface of an immobilization carrier, or immobilizing the fusion protein and uricase (UOX) onto the enzyme membrane surface of an immobilization carrier, and then immobilizing them onto an electrode.

[0039] In one embodiment, the immobilization carrier is cellulose.

[0040] Preferably, the enzyme membrane is a cellulose membrane.

[0041] Preferably, the biosensing element uses a cellulose membrane as a fixed carrier membrane.

[0042] In one embodiment, the electrode may be made of electrode materials commonly used in the art.

[0043] In one embodiment, the biosensing element is a paper-based sensor or a polymer membrane-based sensor.

[0044] On the other hand, this application also provides a uric acid detection kit, the kit comprising the fusion protein and / or the biosensing element described above.

[0045] In one embodiment, the kit may further include a sample carrier and / or a sample collector, which may be existing medical or laboratory equipment.

[0046] In one embodiment, the test samples of the test kit include samples derived from food or samples derived from organisms.

[0047] In one embodiment, the test samples of the test kit are derived from living organisms, including cells, tissues, and bodily fluids.

[0048] In one embodiment, the test sample of the test kit is a biological fluid, including blood, urine, tissue fluid, and lymph.

[0049] In one embodiment, the test sample for the test kit is blood or urine.

[0050] The present invention has the following beneficial effects:

[0051] This application constructs a fusion protein of uric acid oxidase UOX and carbohydrate binding module CBM. The fusion protein not only retains the substrate catalytic ability of UOX, but also has a good binding ability with cellulose. It can be made into a biosensor element and shows a lower detection limit and a wider detection range when used for uric acid detection. It also has good anti-interference ability and stability.

[0052] The sequence information is as follows:

[0053]

[0054] Attached Figure Description

[0055] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0056] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In the drawings:

[0057] Figure 1 The structures of wild-type UOX and fused UOX (CBM3-UOX, CBM3-UOX, and UOX-CBM2) monomers and tetramers were simulated using AlphaFold, and the stability of the enzymes was detected using Ramachandran plots and SDS-PAGE. (A) UOX; (B) CBM3-UOX; (C) UOX-CBM3; (D) UOX-CBM2. M represents the protein standard; uricase / C indicates the result after centrifugation of uricase mixed with cellulose for 30 min; uricase / W indicates the result after centrifugation of uricase mixed with water containing dissolved cellulose for 30 min.

[0058] Figure 2 SDS-PAGE was used to detect the stability of enzymes stored at room temperature for 3 days; (A) UOX-CBM2; (B) UOX-CBM3; (C) CBM3-UOX and mass spectrometry analysis were performed. M represents the protein standard; enzyme / D3 / C indicates the result of urate oxidase after being stored at room temperature for 3 days, mixed with cellulose for 30 min, centrifuged, and the supernatant was collected; enzyme / D3 / W indicates the result of urate oxidase after being stored at room temperature for 3 days, mixed with water containing dissolved cellulose for 30 min, centrifuged, and the supernatant was collected.

[0059] Figure 3 The optimized structure of CBM3-UOX; (A) The monomeric and tetrameric structures of CBM3-Δ7aa-UOX simulated by AlphaFold, and the enzyme binding was detected by Ramachandran plots and SDS-PAGE; (B) The RMSD, RMSF and enzyme-cellulose binding energy of CBM3-Δ10aa-UOX and CBM3-UOX calculated based on kinetic simulation; (C) The monomeric and tetrameric structures of CBM3-Δ10aa-UOX simulated by AlphaFold, and the enzyme binding was detected by Ramachandran plots and SDS-PAGE. Urate oxidase / C and urirate oxidase / W represent the results of enzymes taken directly from the refrigerator, mixed with cellulose or water for 30 min, centrifuged, and the supernatant collected; urirate oxidase / D3 / C and urirate oxidase / D3 / W represent the results of enzymes placed at room temperature for 3 days, mixed with cellulose or water for 30 min, centrifuged, and the supernatant collected.

[0060] Figure 4Enzyme activity stability analysis and analysis of the reasons for the decrease in CBM3-UOX and CBM3-Δ10aa-UOX enzyme activities; (A) Enzyme activity stability of wild-type UOX and fusion enzyme; (B) Uric acid molecule at the active site of wild-type UOX. Nearby amino acid residues; (C)CBM3-UOX active site uric acid molecule Nearby amino acid residues; (D)CBM3-Δ10aa-UOX active site uric acid molecule Nearby amino acid residues.

[0061] Figure 5 SEM images of cellulose membranes modified with different urate oxidases and schematic diagrams of the biosensor fabrication process using wild-type UOX and CBM-fused UOX as sensing elements; (A) Cellulose membrane without modified enzyme molecules; (B) UOX; (C) CBM3-UOX; (D) CBM3-Δ10aa-UOX; (E) CBM3-Δ7aa-UOX; (F) UOX-CBM3; (G) UOX-CBM2; (H) Schematic diagram of enzyme electrode fabrication.

[0062] Figure 6 Cyclic voltammetry tests of electrodes modified with different UOXs; (A) wild-type UOX; (B) CBM3-UOX; (C) CBM3-Δ10aa-UOX; (D) CBM3-Δ7aa-UOX; (E) UOX-CBM3; (F) UOX-CBM2.

[0063] Figures 7A1-7H Electrochemical test results for electrodes modified with different UOXs are shown; among them, Figures 7A1-7D1 The current-time response curves of UOX (A1), CBM3-UOX (B1), CBM3-Δ7aa-UOX (C1), or CBM3-Δ10aa-UOX (D1) modified electrodes to different concentration gradients (0–1.25 mM) of uric acid dissolved in PBS solution (pH 8.0) at a voltage of +0.6 V are shown. Figures 7A2-7D2 Corresponding to Figures 7A1-7D1 Linear correlation analysis between daily uric acid concentration and current difference; Figure 7E The results of three repeated measurements of uric acid at different concentrations (0–1.25 mM) in PBS solution (pH 8.0) using the CBM3-Δ10aa-UOX modified electrode are shown. Figure 7F To Figure 7E RSD of three repeated measurements of uric acid at the same concentration; Figure 7G To detect the selectivity of the CBM3-Δ10aa-UOX modified electrode by mixing uric acid with interfering substances such as glucose, ascorbic acid (AA), dopamine (Dop), and urea; Figure 7H To Figure 7G The current change was calculated after adding interfering elements.

[0064] Figure 8 Analysis of the mechanism by which the current of the uric acid oxidase sensor increases initially, reaches its maximum value, and then gradually decreases; (A) Polymer morphology of wild-type UOX and CBM3-Δ10aa-UOX in solution detected by Native-PAGE; (B) Schematic diagram of possible morphological changes of dimer and tetramer CBM3-Δ10aa-UOX immobilized on cellulose membrane; (C) Schematic diagram of dimer wild-type UOX and CBM3-Δ10aa-UOX forming tetramers and immobilizing on cellulose membrane; (D) Current response curves of a mixed enzyme modified electrode of UOX and CBM3-Δ10aa-UOX at +0.6V to different concentrations of uric acid in PBS solution (pH 8.0) from 0 to 1.25 mM for 7 consecutive days; (E) Current response curves of a mixed enzyme modified electrode of UOX and CBM3-Δ10aa-UOX at +0.6V to different concentrations of uric acid in PBS solution (pH 8.0) from 0 to 1.25 mM for 34 consecutive days. Detailed Implementation

[0065] To more clearly illustrate the overall concept of this application, a detailed description is provided below by way of embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of this application. However, it will be apparent to those skilled in the art that this application can be implemented without one or more of these details. In other instances, to avoid confusion with this application, some technical features well-known in the art have not been described.

[0066] Example 1: Computational Design and Construction of a Highly Stable CBM-Integrated UOX

[0067] In this embodiment, CBMs from the CBM2 and CBM3 families, which have strong binding specificity to cellulose, were selected from the carbohydrate active enzyme database (CAZy, http: / / www.cazy.org / ) and fused with uricase UOX. Specifically, wild-type CBM2 from family 2 is derived from *Thermobifida fusca*, with its coding gene sequence shown in SEQ ID No. 1 and amino acid sequence shown in SEQ ID No. 2; wild-type CBM3 from family 3 is derived from *Acetivibrio thermocellus*, with its coding gene sequence shown in SEQ ID No. 3 and amino acid sequence shown in SEQ ID No. 4; and wild-type UOX (WP_111904949.1) is derived from *Arthrobacter globiformis*, with its coding gene sequence shown in SEQ ID No. 7 and amino acid sequence shown in SEQ ID No. 8.

[0068] 1. CBM-integrated UOX structural simulation and stability assessment

[0069] To evaluate the structural stability of fusion UOXs with different types of CBM (CBM2, CBM3) and different linkage sequences (N-terminus or C-terminus), the monomeric structure of the fusion enzyme was first predicted using ColabFold v1.5.5:AlphaFold2, and the structure with the highest pLDDT score was saved in PDB format. The tetramer structure of the fusion enzyme was then predicted using AlphaFold3, and the confidence level of the tetramer structure was characterized by the pTM parameter. Prediction results for the three fusion enzymes CBM3-UOX, UOX-CBM3, and UOX-CBM2 showed that CBM3-UOX had the highest pLDDT and pTM values. Figure 1 The results (AD) indicate that the structure of CBM3-UOX is more reasonable, and the fusion effect of CBM3 at the N-terminus is better than that at the C-terminus. The amino acid sequences of CBM3-UOX, UOX-CBM3 and UOX-CBM2 are shown in SEQ ID No. 11, SEQ ID No. 12 and SEQ ID No. 13, respectively.

[0070] The PDB files of the three fusion enzymes were further imported into the SAVESv6.0 online website (https: / / saves.mbi.ucla.edu / ), and their stereochemical structural stability was calculated using the built-in PROCHECK simulation program. The results showed that 0.8%, 1.2%, 1.7%, and 1.6% of the residues in the UOX, CBM3-UOX, UOX-CBM3, and UOX-CBM2 structures were located in the essentially allowed and disallowed regions, respectively. Figure 1This indicates that the unstable structural regions increase after UOX is fused with CBM, especially in UOX-CBM3 and UOX-CBM2. Furthermore, the unstable amino acids are mainly located at the N-terminus Asn(N3) of UOX in UOX-CBM3 and UOX-CBM2, or N163 in CBM3-UOX, and at the N-terminus Val(V3), Ser(S4), Asn(N6) of CBM3 in CBM3-UOX, or V305, S306, and N308 in UOX-CBM3. To further verify the accuracy of the prediction results, wild-type UOX and fused UOX were heterologously expressed.

[0071] 2. Enzyme expression plasmids were transformed into E. coli to enrich plasmids.

[0072] The UOX, CBM3-UOX, UOX-CBM3, and UOX-CBM2 genes were ligated to the 5'NdeI and 3'NotI restriction sites of the pET30a(+) expression vector using homologous recombination. The recombination products were mixed with *E. coli* DH5α, incubated on ice for 25 min, heat-shocked for 90 s, and then plated onto 100 μg / mL kanamycin-resistant LB agar plates and incubated overnight at 37°C. Single colonies were picked, and plasmids were extracted for electrophoresis and detection. The target fragments were detected by restriction enzyme digestion, and the plasmids were stored at -20°C.

[0073] 3. Enzyme expression plasmids are transformed into expression hosts and expression is induced.

[0074] Remove E. coli BL21 competent cells from the -80℃ freezer and place them on ice. Transfer 100 μL of competent cells and 1 μL of enzyme expression plasmid into a sterile centrifuge tube, mix gently, and incubate on ice for 25 min. Heat shock at 42℃ for 45 s, place on ice for 2 min, add 700 μL of LB medium to the centrifuge tube, and incubate at 37℃ for at least 1 hour. Centrifuge at 5000 rpm for 1 min, retain about 100 μL of supernatant, gently mix with a pipette, spread on 100 μg / mL kanamycin-resistant LB agar plates, and incubate upside down at 37℃ overnight.

[0075] Ten single clones were picked and cultured overnight at 37°C and 200 rpm in 5 mL test tubes. They were then transferred at a 1:50 volume ratio to 250 mL of fresh liquid LB medium in 500 mL Erlenmeyer flasks and cultured at 37°C until the OD value reached 0.6-0.8. The control group received no IPTG induction, while the experimental group received 1 mM IPTG for induction. The cells were induced at 20°C for 20 h, centrifuged at 8000 rpm and 4°C for 10 min, and then resuspended twice with 0.2 M phosphate buffer (pH 7.4). The cells were then sonicated, placed on ice, and centrifuged at 11000 rpm and 4°C for 10 min. The supernatant was collected and filtered for sterilization. SDS-PAGE and enzyme activity assays were used to detect the expression of the exogenous gene.

[0076] 4. Enzyme molecule isolation, purification, and property determination

[0077] Crude enzyme solutions (UOX, CBM3-UOX, UOX-CBM3, UOX-CBM2) showing active target protein expression as detected by SDS-PAGE were mixed with nickel-containing packing material and subjected to shaking at 10℃ and 100 rpm for 2 h. The target proteins were then eluted with a gradient of imidazole solutions at concentrations of 5 mmol / L, 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 100 mmol / L, and 250 mmol / L. The optimal elution concentrations for UOX, CBM3-UOX, UOX-CBM3, and UOX-CBM2 were 50 mmol / L, 60 mmol / L, 60 mmol / L, and 30 mmol / L, respectively. The UOX, CBM3-UOX, UOX-CBM3, and UOX-CBM2 protein solutions eluted by imidazole were ultrafiltered using a 3K ultrafiltration tube and pH 8.0 phosphate buffer at 4900 rpm and 4°C until the pH of the filtrate was 8.0. Ultrafiltration was then stopped, and the target enzyme molecules obtained by ultrafiltration were collected.

[0078] The target enzyme molecule was detected using SDS-PAGE, and the results are as follows: Figure 1 As shown in AD, the molecular weights of CBM3-UOX, UOX-CBM3, and UOX-CBM2 are significantly larger than that of wild-type UOX. The purified enzymes (UOX, CBM3-UOX, UOX-CBM3, and UOX-CBM2) were mixed with cellulose (UOXs / C) or an aqueous solution of dissolved cellulose (UOXs / W), and the supernatant was obtained by centrifugation. SDS-PAGE analysis showed that wild-type UOX could not bind to cellulose, while the CBM fusion enzymes (CBM3-UOX, UOX-CBM3, and UOX-CBM2) could bind to cellulose, indicating that CBM3 and CBM2 had been successfully fused to the UOX enzyme molecule and maintained their cellulose-binding ability.

[0079] In the CBM3-UOX electrophoresis pattern, additional protein bands of different molecular weights were observed besides the target band. To determine whether these bands were generated by the fusion enzyme cleavage, the fusion enzymes (CBM3-UOX, UOX-CBM3, and UOX-CBM2) were placed at room temperature for 3 days, and the changes in protein bands were then examined. The results showed that the target bands of UOX-CBM3 and UOX-CBM2 completely disappeared, and two additional protein bands with the same molecular weight as UOX and CBM3 or CBM2 appeared. Figure 2 A and B) indicate that UOX-CBM3 and UOX-CBM2 are completely broken at the CBM and UOX linker peptides. After 3 days at room temperature, CBM3-UOX also showed protein band breakage. Further mass spectrometry analysis of different protein bands showed that the breakage points were mainly located at the N-terminus of CBM3 (V3, S4, N6) and the N-terminus of UOX at the linker peptide (N163), but the target protein was still preserved to a certain extent. Figure 2 C). This also shows that the CBM3-UOX structure is more stable and the fusion effect of CBM at the N end is better than that at the C end, which is consistent with the calculation simulation results.

[0080] 5. Optimization of CBM3-UOX sequence structure improves enzyme stability.

[0081] Based on the above analysis and sequence alignment of CBM3 in the CAZy database, we found that most CBMs in the characteristic sequences of the CBM3 family frequently lack N-terminal residues such as P2, V3, S4, G5 and C-terminal residues such as K158, E159, P160, including unstable residues predicted using the CBM3 N-terminus. Therefore, residues such as P2, V3, S4, G5 at the N-terminus and K158, E159, P160 at the C-terminus of CBM3 were deleted and named CBM3-Δ7aa, whose coding gene sequence is shown in SEQ ID No. 5 and whose coding amino acid sequence is shown in SEQ ID No. 6. This was then fused with the N-terminus of UOX to construct the fusion enzyme CBM3-Δ7aa-UOX, whose coding amino acid sequence is shown in SEQ ID No. 14. Using the methods described in steps 2-4 above, the CBM3-Δ7aa-UOX target protein was obtained, and its fragmentation was further examined after being placed at room temperature for 3 days. The results showed that the integrity of the fusion protein increased, indicating that the stability of the fusion enzyme increased after optimizing the CBM3 sequence, but some fragmented bands still remained. Figure 3A). Based on the above analysis, the amino acid at N156 of the N-terminus of the linker peptide UOX in CBM3-Δ7aa-UOX may affect structural stability. Therefore, amino acids M154, S155, and N156 of the N-terminus of UOX in CBM3-Δ7aa-UOX were deleted (i.e., M1, S2, and N3 of the wild-type UOX). The optimized UOX was named UOX-Δ3aa, and its encoding gene sequence is shown in SEQ ID No. 9, while its encoding amino acid sequence is shown in SEQ ID No. 10. The obtained fusion enzyme was named CBM3-Δ10aa-UOX, and its encoding amino acid sequence is shown in SEQ ID No. 15.

[0082] The structural parameters of CBM3-UOX and CBM3-Δ10aa-UOX along their MD trajectories were compared using molecular dynamics (MD) simulations: RMSD, RMSF, and cellulose binding energy. The results are as follows: Figure 3 As shown in Figure B, CBM3-UOX reaches equilibrium with an RMSD of approximately 1.5 nm at about 70 ns, while CBM3-Δ10aa-UOX reaches equilibrium with an RMSD of approximately 0.8 nm at about 20 ns. Generally, a smaller RMSD indicates a more stable protein structure, suggesting that optimization improves the stability of CBM3-Δ10aa-UOX. Furthermore, in CBM3-UOX, the maximum RMSF value of amino acid residues in CBM is approximately 1.5 nm, while the RMSF value of CBM3-Δ10aa-UOX is less than 0.5 nm. Additionally, the number of residues with large mobility ranges is significantly reduced in CBM3-Δ10aa-UOX. These results indicate that deleted residues may have a significant impact on the flexibility and stability of the fused UOX. To investigate whether the reduction in flexibility affected the binding of enzyme molecules to cellulose, the results showed that the binding energies of CBM3-UOX and CBM3-Δ10aa-UOX to cellulose were similar (~-40 Kcal / mol), thus it was speculated that the optimization of the enzyme molecule sequence did not affect the cellulose binding ability.

[0083] Using the methods described in steps 2-4 above, recombinant CBM3-Δ10aa-UOX was obtained. SDS-PAGE analysis before and after storage at room temperature for 3 days showed that the stability of the optimized CBM3-Δ10aa-UOX was significantly improved; no additional bands were detected besides the target enzyme, and CBM3-Δ10aa-UOX could completely bind to cellulose. Figure 3 C). This result is consistent with the MD simulation results, reflecting that the engineering CBM fusion UOX, especially CBM3-Δ10aa-UOX, has higher stability.

[0084] 6. Stability analysis of wild-type UOX and CBM fusion UOX enzyme activities

[0085] Further analysis was conducted on the changes in enzyme activity of modified UOX. Enzyme activity was measured at 2h, 4h, 6h, 8h, and 10h after incubation under biosensor application conditions (24℃, pH 8.0). The activity stability of wild-type UOX and five fusion UOX enzymes (CBM3-UOX, UOX-CBM3, UOX-CBM2, CBM3-Δ7aa-UOX, CBM3-Δ10aa-UOX) was also determined. Figure 4 As shown in Figure A, all UOXs exhibited good enzyme activity stability, which is beneficial for stable uric acid catalysis. However, the enzyme activities of CBM3-UOX and CBM3-Δ10aa-UOX were approximately 75% and 25% of those of UOX, respectively. To investigate the reasons for the decreased enzyme activities of CBM3-UOX and CBM3-Δ10aa-UOX, the active sites of UOX, CBM3-UOX, and CBM3-Δ10aa-UOX were analyzed using uric acid molecules. Analysis of nearby amino acid residues revealed ( Figure 4 (BD) CBM3-UOX and CBM3-Δ10aa-UOX affect the interactions between O2, uric acid molecules, and catalytic residues, thereby reducing enzyme activity. Generally, increased enzyme stability is usually accompanied by decreased enzyme activity, but the enzyme activity of CBM3-Δ10aa-UOX is sufficient for sensing detection requirements, while improved stability is a more desirable performance characteristic for sensing enzymes.

[0086] Example 2: Fabrication of different UOXs sensing elements

[0087] 1. Morphological characterization of cellulose membranes modified with different UOXs

[0088] Cellulose membranes were punched into "O"-shaped sheets using a puncher and adhered to rubber rings to create enzyme membrane rings. Wild-type UOX and CBM-fused UOX (CBM3-UOX, UOX-CBM3, UOX-CBM2, CBM3-Δ7aa-UOX, CBM3-Δ10aa-UOX) at the same concentration (0.1 mg / mL) were added dropwise to the surface of the enzyme membrane rings. After drying at room temperature, the enzyme membrane rings were soaked in deionized water for 24 hours, with the deionized water replaced three times to rinse away loosely bound UOX. The enzyme membrane rings were then dried again at room temperature. The morphology of the UOX-modified cellulose membrane was analyzed by SEM to determine whether UOX was immobilized on the cellulose membrane. The results showed that compared with the bare cellulose membrane without enzyme addition (…),… Figure 5 A), the morphology did not change significantly after the addition of wild-type UOX. Figure 5B) indicates that wild-type UOX cannot be directly immobilized on cellulose membranes without a cross-linking agent. However, when CBM-fused UOX (CBM3-UOX, UOX-CBM3, UOX-CBM2, CBM3-Δ7aa-UOX, CBM3-Δ10aa-UOX) are added to the cellulose membrane, some spots or clusters of enzymes can be clearly observed on the cellulose chains. Figure 5 C-5G) indicates that CBM-fused UOX (CBM3-UOX, UOX-CBM3, UOX-CBM2, CBM3-Δ7aa-UOX, CBM3-Δ10aa-UOX) are directly attached to the cellulose membrane through a specific interaction between CBM and cellulose, and are successfully immobilized. Although the diameter of approximately [missing information] cannot be seen from SEM images, [missing information]. The single urate oxidase molecule, but the aggregation points or clusters observed on cellulose and preliminary evidence of the successful immobilization of fused UOX.

[0089] 2. Fabrication of sensing elements modified with different UOXs

[0090] Mix 5 μL of wild-type UOX enzyme solution with 86 μL of phosphate buffer, 3 μL of 20% bovine serum albumin solution (BSA), and 6 μL of 2% glutaraldehyde, and drop the mixture onto an enzyme membrane ring made of cellulose membrane. Figure 5 H). 100 μL of fusion enzyme solutions of CBM3-UOX, CBM3-Δ7aa-UOX, CBM3-Δ10aa-UOX, UOX-CBM3, and UOX-CBM2 were directly added dropwise onto an enzyme membrane ring made of cellulose membrane. After drying at room temperature for 4 h, the enzyme membrane ring was fixed onto an electrode for performance testing. Figure 5 H). A traditional three-electrode system, the CHI760D electrochemical workstation, was used, with a 3 mm diameter platinum (Pt) electrode as the working electrode, Pt as the counter electrode, and silver / AgCl (3MKCI) as the reference electrode for electrochemical testing.

[0091] Example 3: Electrochemical detection performance characterization of different UOXs sensing elements

[0092] 1. Cyclic Voltammetry (CV) Response Performance

[0093] The electrocatalytic activity of immobilized UOX was measured by CV, with the working electrode potential set between -0.2 V and 0.8 V, and the scan rate at 50 mV / s. -1The CV response curves of different UOX sensing elements to gradient concentrations of uric acid (0.125, 0.25, 0.375, 0.5, 0.625, 0.75, 0.875, 1.0, 1.125, 1.25 mmol / L) were recorded. The results showed that for all electrodes, the oxidation current increased gradient and the reduction current decreased gradient with increasing uric acid concentration in the potential range of +0.2V to +0.8V. Furthermore, a significant H2O2 redox peak was observed at +0.6V, confirming that all UOX were successfully immobilized on the electrodes and maintained their electrocatalytic activity for uric acid. Figure 6 AF).

[0094] 2. Current-time IT response performance

[0095] To further quantitatively evaluate the sensing performance of the UOXs biosensor, the response of the sensing element to gradient concentrations (0–1.25 mM) of uric acid was detected for several consecutive days under +0.6 V conditions using current-time response curves (it). Figure 7A1 As shown in Figure -D1, the test current of the UOX, CBM3-UOX, CBM3-Δ7aa-UOX, and CBM3-Δ10aa-UOX modified electrodes increased with increasing uric acid concentration on the same day. Figures 7A1-7D1 The linear correlation between uric acid concentration and the obtained current difference (ΔI) was calculated, and it can be seen that the correlation coefficient (R) is... 2 Using a performance index of >0.99, the biosensors modified with UOX, CBM3-UOX, CBM3-Δ7aa-UOX, and CBM3-Δ10aa-UOX have lifespans of 3, 6, 7, and 20 days, respectively. Figures 7A2-7D2 This further reflects that the stability of the N-terminal CBM-fused UOX modified bioelectrode is superior to that of wild-type UOX, with CBM3-Δ10aa-UOX exhibiting the best stability. For the C-terminal CBM-fused UOX, a stable current-it curve could not be obtained. This may be because, as described above, CBM-CBM2 and UOX-CBM3 are prone to breakage with UOX, leading to UOX detachment and the inability to form a stable current response.

[0096] according to Figures 7A1-7D2 It can be seen that the bioelectrode modified with CBM3-Δ10aa-UOX exhibits the best stability, showing good linearity within the range of 0–0.625 mM uric acid, and achieving R within 20 days. 2 >0.99. The sensitivity and limit of detection (LOD) of the CBM3-Δ10aa-UOX biosensor on day 20 were 2.13 × 10⁻⁶. 3 μA.mol -1 .L.cm -2And 0.010mM (S / N=3), which is better than the sensitivity of the UOX biosensor on day 5 (0-0.375mM, 1.26×10). 3 μA.mol -1 .L.cm -2 The sensitivity and LOD (0-0.50mM, 1.58×10⁻⁶ mM, S / N=3) of the CBM3-UOX biosensor on day 6 were 0.014mM (S / N=3). 2 μA.mol -1 .L.cm -2 ,0.106mM (S / N=3)), LOD of CBM3-Δ7aa-UOX biosensor on day 7 (0-0.75mM, 2.18×10 3 μA.mol -1 .L.cm -2 (0.012 mM (S / N = 3)). These results demonstrate the advantages of CBM3-Δ10aa-UOX in the determination of low concentrations of uric acid in real serum samples compared to UOX, CBM3-UOX, and CBM3-Δ7aa-UOX.

[0097] 3. Repeatability and selectivity (anti-interference ability)

[0098] The repeatability and selectivity (anti-interference ability) of the CBM3-Δ10aa-UOX biosensor, which exhibited the most stable electrochemical detection performance, were further tested. The reproducibility of the CBM3-Δ10aa-UOX biosensor's response to different concentration gradients of uric acid (0–1.25 mM) was assessed by three repeated measurements. The results showed that the biosensor exhibited almost identical responses in the three repeated measurements. Figure 7E The calculated RSD (Restricted Segment Determination) for three measurements under the same concentration conditions showed a result of 0.4% ≤ RSD ≤ 2.5%, confirming its high repeatability. Figure 7F Selectivity is an important parameter for biosensors because common bioactive substances in blood that coexist with UA, such as glucose (Glu), ascorbic acid (AA), dopamine (Dop), and urea (Urea), can interfere with the current response of UA. This was achieved by detecting a standard UA solution (0.5 mmol / L). -1 The study investigated the effects of common interfering substances in blood samples on the current response of blood samples mixed with interfering substances (AA 100 μmol / L, Dop 200 μmol / L, Glu 8.3 mmol / L, Urea 36 mmol / L) under normal or higher physiological conditions. Results are as follows: Figure 7G , 7HAs shown, the response currents after adding interfering substances were 94.6%, 97.4%, 96.2%, and 97.2% of the standard UA response current, respectively. This indicates that the impact of these potential interferences on the detection of UA in blood samples is very small and negligible.

[0099] 4. The co-fixation of CBM3-Δ10aa-UOX with UOX further improves stability.

[0100] During daily it current monitoring of electrodes modified with different UOXs fusion enzymes, it was observed that the current increased in the first few days, reached a maximum, and then gradually decreased. Figure 7A1 -D1). We hypothesize that interactions between UOX polymers may cause the breakage of the linker peptide between UOX and CBM, thereby affecting the immobilized morphology and exposure of active sites of the UOX polymer. Native-PAGE was used to detect the polymer morphology of wild-type UOX and CBM3-Δ10aa-UOX in solution. The results showed that the natural forms of wild-type UOX and CBM3-Δ10aa-UOX were mainly dimers and tetramers (D1). Figure 8 A). Therefore, as Figure 8 As shown in Figure B, CBM3-Δ10aa-UOX is immobilized on the cellulose membrane in both dimer and tetramer forms. However, CBM3 in the CBM3-Δ10aa-UOX tetramer (molecular 1) can bind to cellulose chains in different directions, creating tensile forces that lead to the breakage of linker peptides, forming molecular morphologies similar to molecular 1'. This promotes molecular motion or exposure of active sites, which may contribute to the increase in current. Furthermore, the interaction forces between dimers (molecular 2, molecular 3) to form tetramers also easily lead to the breakage of linker peptides, forming molecular 2' and molecular 3'-like morphologies, further promoting the current increase phenomenon described in the tetramer. Therefore, we hypothesize that if wild-type UOX is combined with CBM3-Δ10aa-UOX, the dimer wild-type UOX and CBM3-Δ10aa-UOX will tend to form tetramers (molecular 2', molecular 3'), such as... Figure 8 As shown in C, this may reduce the intermolecular tension after fixation, improving sensor stability. Finally, electrochemical detection results confirmed this hypothesis. The current response curves were almost identical during the first 7 days. Figure 8 D) and maintained good catalytic activity until day 34, with a linear range from 0 to 0.875 mmol / L (R). 2 >0.993, 2.26×10 3 μA.mol -1 .L.cm -20.015mM (S / N = 3) Figure 8 E). This further reflects that the enhanced stability of the CBM3-Δ10aa-UOX linker peptide compared to CBM3-UOX and CBM3-Δ7aa-UOX prolongs the current-increasing process, thereby improving the working life. Simultaneously, in the fabrication and application of uric acid sensors, co-immobilizing a modified electrode with a mixture of CBM3-Δ10aa-UOX and wild-type UOX can further improve uric acid sensing performance.

[0101] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A fusion protein, characterized in that, The fusion protein includes a carbohydrate-binding module and uricase, and the amino acid sequence of the fusion protein is shown in any one of SEQ ID NO. 11-15.

2. The use of the fusion protein of claim 1 in the preparation of biosensing elements or detection kits.

3. A method for preparing the fusion protein of claim 1, characterized in that, The method includes the following steps: (a) Transforming a nucleic acid molecule encoding the fusion protein of claim 1 into a microorganism to obtain a recombinant engineered bacterium; (b) Cultivate the recombinant engineered bacteria to express the fusion protein of claim 1.

4. A biosensing element, characterized in that, The biosensing element contains the fusion protein as described in claim 1.

5. The biosensing element according to claim 4, characterized in that, The biosensing element contains the fusion protein and urate oxidase as described in claim 1.

6. A method for preparing the biosensing element according to any one of claims 4-5, characterized in that, The method includes immobilizing the fusion protein of claim 1 onto the enzyme membrane surface of the immobilization carrier, or immobilizing the fusion protein of claim 1 and uric acid oxidase onto the enzyme membrane surface of the immobilization carrier, and then immobilizing them onto the electrode.

7. The method according to claim 6, characterized in that, The fixation carrier is cellulose.

8. A uric acid detection kit, characterized in that, The kit includes the fusion protein of claim 1 and / or the biosensing element of any one of claims 4-5.

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