Fused urate oxidase and application thereof in biosensing element

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology, and specifically relates to a fusion protein constructed by connecting a uricase gene with a carbohydrate binding domain gene, and application of the fusion protein in a biosensor element. Background Art

[0002] Uric acid (UA) is an important nitrogen-containing compound that is commonly found in biological fluids such as serum and urine. It is the main end product of purine metabolism. The normal levels of serum uric acid in healthy people are approximately 0.21-0.43mM and 0.15-0.36mM in adult males and adult females, respectively, while the uric acid concentration in gout patients can reach 1.49-4.46mM. Abnormal uric acid levels in biological fluids can indicate diseases such as gout, metabolic syndrome and kidney disease. Therefore, uric acid is an effective biomarker for detecting purine metabolism disorders. Establishing a method for rapid and reliable detection of uric acid concentrations in biological fluids is of great significance for the diagnosis and treatment of diseases.

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

[0004] In previous studies, different types of enzyme immobilization techniques have been used to construct amperometric UA biosensors. Physical adsorption and embedding are commonly used methods to immobilize UOX, but the binding force of adsorption is weak and easily affected by environmental changes, which reduces the operation and storage stability of the biosensor. UOX encapsulation is usually achieved by polymers such as polyaniline (PANi) and polypyrrole, but the leakage of immobilized enzymes from the polymers poses a challenge to their stability. The method of 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) is also a commonly used method for chemically immobilizing UOX, which obviously avoids enzyme desorption and leakage, but the extensive enzyme inactivation and low reproducibility caused by covalent bonds are the main limitations of these methods in the application of biosensors. In recent years, affinity adsorption has been proven to be a promising enzyme immobilization strategy that can improve the performance of enzyme biosensors.

[0005] The carbohydrate binding domain (CBM) is a continuous amino acid sequence in carbohydrate-active enzymes and is a type of non-catalytic domain with an independent folding structure. The CBM is connected to the catalytic domain through a linker and plays an important role in the recognition and directional binding of enzyme molecules to substrates. It has binding specificity for different types of polysaccharides, especially cellulose. With the development of molecular biology, synthetic biology, computer-aided simulation and other technologies, researchers are currently using fusion DNA technology to try to obtain a variety of CBM and enzyme fusion proteins to improve the affinity between enzyme molecules and substrates, enzyme molecule stability or enzyme activity.

[0006] In the previous work, the applicant successfully constructed a biosensor element based on the specific binding of CBM and cellulose, such as patents CN113061189B and CN112851821B, which fused the carbohydrate binding module (CBM2) of the 2 family with glucose oxidase (GOD) and glucose dehydrogenase (GDH) by genetic engineering methods to improve the glucose sensing performance. However, the construction of a biosensor element for detecting uric acid concentration has not been reported at home and abroad. In addition, the structure and catalytic mechanism of UOX are completely different from those of GOD, so how to construct a stable and active CBM-fused UOX as a UA biosensor element is still crucial.

[0007] UOX is a functional tetrameric barrel structure, whose active site is composed of the interface residues of two monomers, and each monomer contains two identical super secondary domains with ββααββ antiparallel structure, called Tunnelling-fold domain. 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. The complementary arrangement of polar groups and hydrophobic groups at the interface of close contact between subunits promotes the binding of specific subunits. In the catalytic process of UOX, a coenzyme-free approach is used to promote and control O2 chemistry, and different catalytic mechanisms similar to flavin-dependent oxidases or flavin-dependent monooxygenases are proposed, but their mechanism of action is not fully understood. Due to the multi-subunit-dependent catalytic activity and unclear catalytic mechanism of UOX, there have been few studies on modified UOX in recent decades. This has brought great challenges to obtaining stable gene fusion UOX. Summary of the invention

[0008] In order to solve the above problems, the present invention aims to provide a fusion protein and its application in a biosensor element.

[0009] In one aspect, the present application provides a fusion protein comprising a carbohydrate binding module (CBM) and urate oxidase (UOX).

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

[0011] Preferably, the CBM3 is an optimized CBM3, and the optimized CBM3 lacks amino acids 1-4 and 157-159 relative to the parent CBM3; the amino acid sequence of the parent 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, and the optimized UOX lacks amino acids 1-3 relative to the parent UOX; the amino acid sequence of the parent 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-terminus or C-terminus of UOX.

[0016] Preferably, the CBM is placed at the N-terminus of the UOX.

[0017] In one embodiment, the amino acid sequence of the fusion protein is shown in any one of SEQ ID NOs. 11-15.

[0018] On the other hand, the present application also provides a biomaterial, wherein the biomaterial comprises any one or more of the following:

[0019] I. a nucleic acid molecule encoding 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. A recombinant microorganism, which contains the nucleic acid molecule described in I or 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 a promoter, a terminator, a marker gene, etc., as long as they can achieve the expression of the nucleic acid molecule.

[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 a plasmid, a phage or a viral vector, preferably a plasmid, 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, the present application also provides the use of the fusion protein in preparing a biosensor element or a detection kit.

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

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

[0029] On the other hand, the present 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 a microorganism to obtain a recombinant engineered bacterium;

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

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

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

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

[0035] On the other hand, the present application also provides a biosensor element, which contains the fusion protein.

[0036] In one embodiment, the biosensor element further comprises urate oxidase (UOX). Preferably, the UOX is a 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 comprises the fusion protein and urate oxidase (UOX).

[0038] On the other hand, the present application also provides a method for preparing the biosensor element, which includes fixing the fusion protein to the enzyme membrane surface of a fixed carrier, or fixing the fusion protein and urate oxidase (UOX) to the enzyme membrane surface of a fixed carrier, and then fixing them to an electrode.

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

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

[0041] Preferably, the biosensor 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 film-based sensor.

[0044] On the other hand, the present application also provides a uric acid detection kit, which includes the fusion protein and / or the biosensor element.

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

[0046] In one embodiment, the test sample of the detection kit includes a sample derived from food or a sample derived from an organism.

[0047] In one embodiment, the detection sample of the detection kit is derived from an organism, including cells, tissues, and biological fluids.

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

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

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

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

[0052] The sequence information is as follows:

[0053]

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

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

[0056] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0057] Figure 1 AlphaFold was used to simulate the structures of wild-type UOX and fusion UOX (CBM3-UOX, CBM3-UOX and UOX-CBM2) monomers and tetramers, and Ramachandran plots and SDS-PAGE were used to detect the stability of the enzymes; (A) UOX; (B) CBM3-UOX; (C) UOX-CBM3; (D) UOX-CBM2. Where M is a protein standard substance; Urate oxidase / C means that urate oxidase is mixed with cellulose for 30 minutes, and the supernatant is taken after centrifugation; Urate oxidase / W means that urate oxidase is mixed with water dissolving cellulose for 30 minutes, and the supernatant is taken after centrifugation.

[0058] Figure 2 .SDS-PAGE detection of enzyme stability after 3-day storage at room temperature; (A) UOX-CBM2; (B) UOX-CBM3; (C) CBM3-UOX and mass spectrometry analysis. M is a protein standard; Enzyme / D3 / C indicates that urate oxidase was placed at room temperature for 3 days and then mixed with cellulose for 30 minutes, and then centrifuged to obtain the supernatant for detection; Enzyme / D3 / W indicates that urate oxidase was placed at room temperature for 3 days and then mixed with water that dissolved cellulose for 30 minutes, and then centrifuged to obtain the supernatant for detection.

[0059] Figure 3 .The structure of optimized CBM3-UOX; (A) The monomer and tetramer structures of CBM3-Δ7aa-UOX simulated by AlphaFold, and the binding of enzymes 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 monomer and tetramer structures of CBM3-Δ10aa-UOX simulated by AlphaFold, and the binding of enzymes detected by Ramachandran plots and SDS-PAGE. Among them, uricase / C and uricase / W represent the results of the enzyme taken out of the refrigerator and directly mixed with cellulose or water for 30 minutes, and the supernatant was taken after centrifugation; uricase / D3 / C and uricase / D3 / W represent the results of the enzyme placed at room temperature for 3 days and mixed with cellulose or water for 30 minutes, and the supernatant was taken after centrifugation.

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

[0061] Figure 5 .SEM detection images of cellulose membranes modified with different urate oxidases and schematic diagram of the preparation process of biosensors 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 preparation.

[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] Figure 7A1-Figure 7H The electrochemical test results of different UOXs modified electrodes; Figures 7A1-7D1 The current-time it 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, respectively; Figures 7A2-7D2 Corresponding to Figures 7A1-7D1 Linear correlation analysis between daily uric acid concentration and current difference; Fig. 7E The results of three repeated measurements of uric acid at different concentrations of 0 to 1.25 mM in PBS solution (pH 8.0) using CBM3-Δ10aa-UOX modified electrode; Figure 7F For Fig. 7E RSD of three repeated determinations of the same concentration of uric acid; Figure 7G To detect the selectivity of CBM3-Δ10aa-UOX modified electrode by mixing uric acid with interferents such as glucose, ascorbic acid (AA), dopamine (Dop), and urea; Figure 7H For Figure 7G The current change calculated after adding the interferent.

[0064] Figure 8 .Analysis of the mechanism of the urate oxidase sensor current increasing at the initial stage and then gradually decreasing after reaching the maximum value; (A) Detection of the polymer morphology of wild-type UOX and CBM3-Δ10aa-UOX in the solution by Native-PAGE; (B) Schematic diagram of the possible morphological changes of dimer and tetramer CBM3-Δ10aa-UOX fixed on the cellulose membrane; (C) Schematic diagram of the formation of tetramers of dimer wild-type UOX and CBM3-Δ10aa-UOX and fixation on the cellulose membrane; (D) Current response curves of the UOX and CBM3-Δ10aa-UOX mixed enzyme modified electrode to different concentrations of uric acid in PBS solution (pH 8.0) from 0 to 1.25 mM at a voltage of +0.6 V for 7 consecutive days; (E) Current response curves of the UOX and CBM3-Δ10aa-UOX mixed enzyme modified electrode to different concentrations of uric acid in PBS solution (pH 8.0) from 0 to 1.25 mM at a voltage of +0.6 V for 34 consecutive days. DETAILED DESCRIPTION

[0065] In order to more clearly explain the overall concept of the application, the following is described in detail by way of embodiment. In the following description, a large amount of specific details are provided so as to provide a more thorough understanding of the application. However, it is obvious to those skilled in the art that the application can be implemented without one or more of these details. In other examples, in order to avoid confusion with the application, some technical features well known in the art are not described.

[0066] Example 1: Computational design and construction of highly stable CBM fusion UOX

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

[0068] 1. CBM fusion UOX structure simulation and stability assessment

[0069] To evaluate the structural stability of fusion UOX with different types of CBM (CBM2, CBM3) and different connection orders (N-terminal or C-terminal), the monomer structure of the fusion enzyme was first predicted by ColabFold v1.5.5: AlphaFold2, and the structure with the highest pLDDT score was saved in PDB format. AlphaFold3 was used to predict the tetramer structure of the fusion enzyme, and the structural prediction confidence parameter pTM was used to characterize the credibility of the tetramer structure. The prediction results of the three fusion enzymes CBM3-UOX, UOX-CBM3 and UOX-CBM2 showed that CBM3-UOX had the highest pLDDT and pTM values ​​( Figure 1 AD), indicating 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 the stereochemical structure 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 structures of UOX, CBM3-UOX, UOX-CBM3 and UOX-CBM2 were located in the basic allowed region and the unallowed region, respectively ( Figure 1AD), which indicates that the unstable structural region increases after UOX is fused to CBM, especially UOX-CBM3 and UOX-CBM2. And the unstable amino acids are mainly located in UOX-CBM3, UOX-CBM2, UOXN-terminal Asn (N3) or N163 of CBM3-UOX, and CBM3-UOX N-terminal Val (V3), Ser (S4), Asn (N6) or V305, S306, N308 in UOX-CBM3. To further verify the accuracy of the prediction results, wild-type UOX and fusion UOX were heterologously expressed.

[0071] 2. Enzyme molecule expression plasmid transformation E. coli enrichment plasmid

[0072] The UOX, CBM3-UOX, UOX-CBM3 and UOX-CBM2 genes were connected to the 5'NdeI and 3'NotI restriction sites of the pET30a(+) expression vector by homologous recombination. The gene recombination products were mixed with Escherichia coli DH5α, placed on ice for 25 minutes, and then heat-shocked for 90 seconds and spread on 100μg / mL kanamycin-resistant LB agar plates and cultured at 37°C overnight. Single colonies were picked, and then the plasmids were extracted for electrophoresis detection, and the target fragments were detected by endonuclease digestion, and the plasmids were stored at -20°C.

[0073] 3. Enzyme molecule expression plasmid is transformed into expression host and induced to express

[0074] Take out the E. coli BL21 competent cells from the -80℃ refrigerator and place them on ice; transfer 100μL of competent cells and 1μL of enzyme molecule expression plasmid into a sterile centrifuge tube, gently mix evenly, and place on ice for 25min; heat shock in a 42℃ water bath for 45s, place on ice for 2min, add 700μL of LB culture medium into the centrifuge tube, and incubate at 37℃ for more than 1 hour; centrifuge at 5000rpm for 1min, retain about 100μL of supernatant, gently blow and mix with a pipette, spread on a 100μg / mL kanamycin resistance LB agar culture plate, and culture inverted overnight at 37℃.

[0075] Pick 10 monoclones into 5mL test tube culture medium, culture overnight at 37℃, 200rpm; then transfer to 500mL Erlenmeyer flask with 250mL fresh liquid LB culture medium at a volume ratio of 1:50, and culture at 37℃ until OD value is 0.6-0.8. The control group was not induced by IPTG, while the experimental group was induced by IPTG with a final concentration of 1mM, induced at 20℃ for 20h, centrifuged at 8000rpm, 4℃ for 10min; resuspended and washed twice with 0.2M phosphate buffer (pH7.4); the bacterial cells were ultrasonically broken, placed on ice, centrifuged at 11000rpm, 4℃ for 10min, and the supernatant was taken and filtered for sterilization. SDS-PAGE and enzyme activity were used to detect the expression of exogenous genes.

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

[0077] The crude enzyme solution (UOX, CBM3-UOX, UOX-CBM3, UOX-CBM2) with active target protein expression detected by SDS-PAGE was mixed with a filler containing nickel and rotated in a shaker at 10°C and 100 rpm for 2 hours. Then, the target protein was gradiently eluted with an imidazole solution with a concentration of 5mmol / L, 10mmol / L, 20mmol / L, 30mmol / L, 40mmol / L, 50mmol / L, 60mmol / L, 100mmol / L, and 250mmol / L. The optimal elution concentrations of UOX, CBM3-UOX, UOX-CBM3, and UOX-CBM2 were 50mmol / L, 60mmol / L, 60mmol / L, and 30mmol / L, respectively. Use 3K ultrafiltration tubes and pH 8.0 phosphate buffer to ultrafilter the UOX, CBM3-UOX, UOX-CBM3, and UOX-CBM2 protein solutions eluted with imidazole at 4900 rpm and 4°C until the pH of the flowing buffer is 8.0. Stop ultrafiltration and collect the target enzyme molecules obtained by ultrafiltration.

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

[0079] In the CBM3-UOX electrophoresis detection spectrum, it was found that in addition to the target bands, there were additional protein bands with molecular weights. In order to detect whether these bands were produced by the cleavage of the fusion enzyme, the fusion enzymes (CBM3-UOX, UOX-CBM3, and UOX-CBM2) were further placed at room temperature for 3 days to detect the changes in protein bands. The results showed that the target bands of UOX-CBM3 and UOX-CBM2 disappeared and two other protein bands with the same molecular weight as UOX and CBM3 or CBM2 appeared ( Figure 2 A and B), indicating that UOX-CBM3 and UOX-CBM2 were all broken at the CBM and UOX connecting peptides. After 3 days at room temperature, CBM3-UOX also showed protein band breakage. Further mass spectrometry detection of different protein bands showed that the breakpoints were mainly located at the CBM3 N-terminal V3, S4, N6) and the UOX N-terminal N163 at the connecting peptide, but the target protein was still retained 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-terminus is better than that at the C-terminus, which is consistent with the computational simulation results.

[0080] 5. CBM3-UOX sequence structure optimization improves enzyme molecular stability

[0081] Based on the above analysis and sequence alignment of CBM3 in the CAZy database, we found that in the characteristic sequences of the CBM3 family, most CBMs often lack residues such as N-terminal P2, V3, S4, G5 and C-terminal K158, E159, P160, including unstable residues predicted by the N-terminal of CBM3. Therefore, the residues P2, V3, S4, G5 and C-terminal K158, E159, P160 of CBM3 were deleted and named CBM3-Δ7aa, the coding gene sequence of which is shown in SEQ ID No.5, the coding amino acid sequence is shown in SEQ ID No.6, and it was fused with the N-terminal of UOX to construct the fusion enzyme CBM3-Δ7aa-UOX, the coding amino acid sequence of which is shown in SEQ ID No.14. The method of steps 2-4 above was used to obtain the CBM3-Δ7aa-UOX target protein and further detect its fragmentation 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 there were still some broken bands ( Figure 3A). Based on the above analysis, the amino acid at N156 at the N-terminus of the connecting peptide UOX in CBM3-Δ7aa-UOX may affect the structural stability, so the amino acids M154, S155, and N156 at the N-terminus of UOX in CBM3-Δ7aa-UOX were deleted (i.e., M1, S2, and N3 at the N-terminus of the wild-type UOX), and the optimized UOX was named UOX-Δ3aa, the coding gene sequence of which is shown in SEQ ID No.9, and the coding amino acid sequence is shown in SEQ ID No.10; the obtained fusion enzyme was named CBM3-Δ10aa-UOX, and the coding amino acid sequence is shown in SEQ ID No.15.

[0082] Through MD simulation, the structural parameters of CBM3-UOX and CBM3-Δ10aa-UOX were compared in the MD trajectory: RMSD, RMSF, and cellulose binding energy. Figure 3 As shown in B, CBM3-UOX reaches an equilibrium RMSD value of about 1.5nm at about 70ns, while CBM3-Δ10aa-UOX can reach an equilibrium RMSD value of about 0.8nm at about 20ns. Generally, the smaller the RMSD, the more stable the protein structure. It is speculated that the stability of CBM3-Δ10aa-UOX is improved after optimization. At the same time, in CBM3-UOX, the maximum RMSF value of the amino acid residues in the CBM is about 1.5nm, while the RMSF value of CBM3-Δ10aa-UOX is less than 0.5nm, and the number of residues with a large range of movement in CBM3-Δ10aa-UOX is significantly reduced. These results indicate that the deleted residues may have a significant impact on the flexibility and stability of the fused UOX. To explore whether the reduction in flexibility affects the binding of the enzyme molecule to cellulose, the results showed that the binding energies of CBM3-UOX and CBM3-Δ10aa-UOX to cellulose were similar (~-40Kcal / mol), so it was inferred that the optimization of the enzyme molecule sequence did not affect the cellulose binding ability.

[0083] The recombinant CBM3-Δ10aa-UOX was obtained by the method of steps 2-4 above. The SDS-PAGE analysis results before and after storage at room temperature for 3 days showed that the stability of the optimized CBM3-Δ10aa-UOX was greatly improved, no additional bands were detected except 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 engineered CBM-fused UOX, especially CBM3-Δ10aa-UOX, has a higher stability.

[0084] 6. Analysis of the enzyme activity stability of wild-type UOX and CBM-fused UOX

[0085] To further analyze the changes in the enzyme activity of the modified UOX, the enzymes were incubated under the biosensor application conditions (24 °C, pH 8.0) and taken out for enzyme activity determination at 2 h, 4 h, 6 h, 8 h, and 10 h, respectively, to determine the activity stability of the wild-type UOX and five fusion UOX enzymes (CBM3-UOX, UOX-CBM3, UOX-CBM2, CBM3-Δ7aa-UOX, CBM3-Δ10aa-UOX). Figure 4 As shown in A, all UOXs showed good enzyme activity stability, which was conducive to the stable catalysis of uric acid. The enzyme activities of CBM3-UOX and CBM3-Δ10aa-UOX were about 75% and 25% of UOX, respectively. In order to explore the reasons for the decrease in the enzyme activities of CBM3-UOX and CBM3-Δ10aa-UOX, the uric acid molecules in the active sites of UOX, CBM3-UOX and CBM3-Δ10aa-UOX were analyzed. The analysis of the nearby amino acid residues revealed that ( Figure 4 BD), CBM3-UOX and CBM3-Δ10aa-UOX affect the interaction between O2, uric acid molecules and catalytic residues, thereby reducing enzyme activity. Generally, increased enzyme molecule stability is usually accompanied by reduced enzyme activity, but the enzyme activity of CBM3-Δ10aa-UOX can meet the requirements of sensor detection, and improved stability is a more desired performance of sensor enzymes.

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

[0087] 1. Characterization of morphology of cellulose membranes modified with different UOXs

[0088] The cellulose membrane was punched into an "O"-shaped sheet using a puncher and glued to a rubber ring to make an enzyme membrane ring. The same concentration (0.1 mg / mL) of wild-type UOX and CBM-fused UOX (CBM3-UOX, UOX-CBM3, UOX-CBM2, CBM3-Δ7aa-UOX, CBM3-Δ10aa-UOX) was dripped onto the surface of the enzyme membrane ring. After drying at room temperature, the enzyme membrane ring was soaked in deionized water for 24 hours, and the deionized water was replaced 3 times in the middle to rinse the UOX that was not firmly bound. The enzyme membrane ring was dried at room temperature again, and the morphology of the UOXs-modified cellulose membrane was detected by SEM to determine whether UOX was fixed to the cellulose membrane. The results showed that compared with the bare cellulose membrane without the addition of enzyme solution ( Figure 5 A), the morphology did not change significantly after adding wild-type UOX ( Figure 5B), indicating that wild-type UOX cannot be directly fixed on the cellulose membrane without a cross-linking agent. When CBM-fused UOX (CBM3-UOX, UOX-CBM3, UOX-CBM2, CBM3-Δ7aa-UOX, CBM3-Δ10aa-UOX) was added to the cellulose membrane, some spots or clusters of enzymes could be clearly observed on the cellulose chains ( Figure 5 C-5G), indicating that CBM-fused UOX (CBM3-UOX, UOX-CBM3, UOX-CBM2, CBM3-Δ7aa-UOX, CBM3-Δ10aa-UOX) was directly attached to the cellulose membrane through the specific interaction between CBM and cellulose and was successfully immobilized on the cellulose membrane. Single uricase molecules were not detected, but aggregation points or clusters were observed on cellulose, which preliminarily demonstrated the successful immobilization of fused UOX.

[0089] 2. Preparation of different UOXs modified sensing elements

[0090] 5 μL of wild-type UOX enzyme solution was mixed with 86 μL of phosphate buffer, 3 μL of 20% bovine serum albumin solution (BSA), and 6 μL of 2% glutaraldehyde, and then dripped onto the enzyme membrane circle made of cellulose membrane ( Figure 5 H). 100 μL of the fusion enzyme solution of CBM3-UOX, CBM3-Δ7aa-UOX, CBM3-Δ10aa-UOX, UOX-CBM3, and UOX-CBM2 was directly dripped onto the enzyme membrane circle made of cellulose membrane, and after drying at room temperature for 4 h, the enzyme membrane circle was fixed to the electrode for performance testing ( Figure 5 H). Electrochemical tests were performed using a conventional three-electrode system CHI760D electrochemical workstation, with 3 mm diameter platinum (Pt) as the working electrode, Pt as the counter electrode, and silver / AgCl (3M KCl) as the reference electrode.

[0091] Example 3: Characterization of electrochemical detection performance 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 a scan rate of 50 mVs -1, recorded the CV response curves of different UOXs sensor 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, 1.25mmol / L). The results showed that in the potential range of +0.2V to +0.8V, the oxidation current of all electrodes increased with the increase of uric acid concentration, and the reduction current decreased with the increase of uric acid concentration. There was an obvious H2O2 characteristic redox peak at +0.6V, which confirmed that all UOXs were successfully fixed on the electrode and maintained the electrocatalytic activity for uric acid ( Figure 6 AF).

[0094] 2. Current-time it response performance

[0095] In order to further quantitatively evaluate the sensing performance of the UOXs biosensor, the current-time response curve was used to detect the response of the sensing element to gradient concentrations of uric acid (0-1.25 mM) at +0.6 V for several consecutive days. Fig.7A1 -D1, the test current of the electrodes modified with UOX, CBM3-UOX, CBM3-Δ7aa-UOX and CBM3-Δ10aa-UOX increased with the increase of uric acid concentration on the same day. Figure 7A1-Figure 7D1 The linear correlation between the uric acid concentration and the current difference (ΔI) was calculated. It can be seen that the correlation coefficient (R 2 )>0.99 as the working performance index, the working life of UOX, CBM3-UOX, CBM3-Δ7aa-UOX and CBM3-Δ10aa-UOX modified biosensors were 3, 6, 7 and 20 days, respectively ( Figure 7A2-Figure 7D2 ), further reflecting that the stability of the N-terminal CBM-fused UOX modified bioelectrode is better than that of the wild-type UOX, and CBM3-Δ10aa-UOX has the best stability. For the C-terminal CBM-fused UOX, a stable current it curve cannot be obtained. This may be because the CBM and UOX of UOX-CBM2 and UOX-CBM3 are easily broken as described above, resulting in the shedding of UOX and the inability to form a stable current response.

[0096] according to Figure 7A1-Figure 7D2 It can be seen that the bioelectrode modified with CBM3-Δ10aa-UOX has the best stability, with a good linear relationship in the range of 0-0.625 mM uric acid, and R 2 >0.99. The sensitivity and limit of detection (LOD) of CBM3-Δ10aa-UOX biosensor on day 20 were 2.13×10 3 μA.mol -1 .L.cm -2and 0.010 mM (S / N = 3), which was better than the sensitivity of the UOX biosensor on day 5 (0-0.375 mM, 1.26 × 10 3 μA.mol -1 .L.cm -2 , 0.014 mM (S / N = 3)), the sensitivity and LOD of CBM3-UOX biosensor on day 6 (0-0.50 mM, 1.58 × 10 2 μA.mol -1 .L.cm -2 , 0.106 mM (S / N = 3)), LOD of CBM3-Δ7aa-UOX biosensor on day 7 (0-0.75 mM, 2.18 × 10 3 μA.mol -1 .L.cm -2 , 0.012 mM (S / N=3)). These results show the advantages of CBM3-Δ10aa-UOX in the determination of low-concentration uric acid in actual serum samples compared with 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 with the most stable electrochemical detection performance were further tested. The reproducibility of the CBM3-Δ10aa-UOX biosensor was tested by repeating the response of the biosensor to different concentration gradients of uric acid (0-1.25mM) three times. The results showed that the biosensor showed almost the same response ( Fig. 7E ), the calculation of the three measurement errors (RSD) under the same concentration conditions showed that 0.4% ≤ RSD ≤ 2.5%, confirming its high repeatability ( Figure 7F Selectivity is an important parameter of biosensors because common physiological electroactive substances coexisting with UA in blood, such as glucose (Glu), ascorbic acid (AA), dopamine (Dop), and urea (Urea), will interfere with the current response of UA. -1 ) and its current response after mixing with interfering substances (AA 100μmol / L, Dop 200μmol / L, Glu 8.3mmol / L, Urea 36mmol / L) under normal or higher physiological conditions, and the influence of common interfering substances in blood samples was explored. Figure 7G , 7HAs shown, the response currents after adding the interferents were 94.6%, 97.4%, 96.2% and 97.2% of the standard UA response currents, respectively, which indicates that the effects of these potential interferences on UA ​​detection in blood samples are very small and can be ignored.

[0099] 4. Mixed immobilization of CBM3-Δ10aa-UOX and UOX further improves stability

[0100] During the daily IT current detection process of electrodes modified with different UOXs fusion enzymes, it can be observed that the current increases in the first few days, reaches the maximum value, and then gradually decreases ( Fig.7A1 -D1). We speculate that the interaction between UOX polymers may cause the cleavage of the connecting peptide between UOX and CBM, thereby affecting the immobilization morphology of UOX polymers and the exposure of active sites. 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 ( Figure 8 A) Therefore, if Figure 8 As shown in B, CBM3-Δ10aa-UOX is immobilized on the cellulose membrane in the form of dimers and tetramers. However, CBM3 in the CBM3-Δ10aa-UOX tetramer (molecular 1) can bind to cellulose chains in different directions to form pulling forces in different directions, resulting in the breakage of the connecting peptide and the formation of a molecular morphology similar to molecule 1', promoting the movement of the molecule or the exposure of the active site, which may have contributed to the increase in current to a certain extent. In addition, the interaction force between dimers (molecular 2, molecule 3) to form a tetramer also easily leads to the breakage of the connecting peptide to form a morphology similar to molecule 2' and molecule 3', further promoting the current increase phenomenon described in the tetramer. Therefore, we speculate that if wild-type UOX is combined with CBM3-Δ10aa-UOX, the dimeric wild-type UOX and CBM3-Δ10aa-UOX will tend to form a tetramer (molecular 2", molecule 3"), as shown in Figure 1. Figure 8 C, which may reduce the intermolecular tension after fixation and improve sensor stability. Finally, the electrochemical detection results confirmed this hypothesis. In the first 7 days, the current response curves were almost the same ( Figure 8 D) and maintained good catalytic activity until the 34th day, with a linear range from 0 to 0.875mmol / L (R 2 >0.993,2.26×10 3 μA.mol -1 .L.cm -2,0.015mM(S / N=3))( Figure 8 E). This further reflects that the stability of the CBM3-Δ10aa-UOX connecting peptide is enhanced compared with that of CBM3-UOX and CBM3-Δ7aa-UOX, which prolongs the current increase process and thus improves the working life. At the same time, in the preparation and use of uric acid sensors for sensing detection, CBM3-Δ10aa-UOX and wild-type UOX can be mixed and co-immobilized to modify the electrode to further improve the uric acid sensing detection performance.

[0101] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A fusion protein, characterized in that The fusion protein includes a carbohydrate binding module (CBM) and urate oxidase (UOX).

2. The fusion protein according to claim 1, characterized in that The amino acid sequence of the CBM is selected from SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.6; the amino acid sequence of the UOX is selected from SEQ ID NO.8 or SEQ ID NO.

10.

3. The fusion protein according to claim 1, characterized in that The amino acid sequence of the fusion protein is shown in any one of SEQ ID NOs. 11-15.

4. Use of the fusion protein according to any one of claims 1 to 3 in the preparation of a biosensor element or a detection kit.

5. A method for preparing the fusion protein according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: (a), transforming a nucleic acid molecule encoding the fusion protein of any one of claims 1 to 3 into a microorganism to obtain a recombinant engineered bacterium; (b) culturing the recombinant engineered bacteria to express the fusion protein according to any one of claims 1 to 3.

6. A biosensor element, characterized in that: The biosensor element contains the fusion protein shown in any one of claims 1-3.

7. The biosensor element according to claim 6, characterized in that: The biosensor element contains the fusion protein shown in any one of claims 1 to 3 and urate oxidase (UOX).

8. A method for preparing the biosensor element according to any one of claims 6 to 7, characterized in that: The method comprises fixing the fusion protein described in any one of claims 1 to 3 to the enzyme membrane surface of a fixed carrier, or fixing the fusion protein described in any one of claims 1 to 3 and urate oxidase (UOX) to the enzyme membrane surface of a fixed carrier, and then fixing them to an electrode.

9. The method according to claim 8, characterized in that The fixed carrier is cellulose.

10. A uric acid detection kit, characterized in that: The kit comprises the fusion protein according to any one of claims 1 to 3 and / or the biosensor element according to any one of claims 6 to 7.

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