Pyranose oxidase mutant and application thereof in glucose detection

Abstract

The invention belongs to the technical field of bioengineering and enzyme engineering, and particularly relates to a pyranose oxidase mutant and application thereof in glucose detection. The invention discloses a pyranose oxidase mutant. The mutant is obtained by mutating the 155th site of an amino acid sequence as shown in SEQ ID NO. 2; according to the mutant, glutamine at the 155th position is mutated into threonine, alanine or glycine. Compared with a wild type, the mutant disclosed by the invention has the advantages that the affinity to interferents such as 1, 5-anhydrous-D-glucitol and the like is remarkably reduced while a high catalytic rate is reserved. The mutant provided by the invention has good linear response in a glucose concentration range of 0.02-5 mM, and especially can accurately detect low-concentration glucose in the presence of interference of physiological concentration 1, 5-AG, so that the problem of false positive caused by interferents in the prior art is effectively solved. The mutant enzyme can be widely applied to the fields of enzymatic biosensors, saliva and tear noninvasive blood glucose monitoring and the like.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering and enzyme engineering technology, and specifically relates to a pyranose oxidase mutant and its application in glucose detection. Background Technology

[0002] Pyranose oxidase (PROD) is a flavin enzyme widely used in biocatalysis and the detection of blood glucose and 1,5-AG. However, while wild-type PROD exhibits good catalytic activity towards glucose, its broad substrate scope is a significant drawback. It is susceptible to interference from structural analogs such as 1,5-anhydrous D-glucanol (1,5-AG) and galactose.

[0003] In human physiological fluids (such as blood), the concentration of 1,5-AG is relatively stable. Especially in non-blood fluids such as saliva and tears, glucose concentrations are usually low (<1 mM), and the presence of interfering substances can lead to a significant decrease in the signal-to-noise ratio. For example, when glucose concentrations are below 1 mM, wild-type PROD often produces significantly inflated readings (overestimations) due to catalysis of 1,5-AG.

[0004] Existing technologies for modifying PROD mainly focus on optimizing active sites, but often face the challenge of an "activity-specificity trade-off," meaning that mutations that increase specificity often lead to a sharp decrease in the maximum reaction rate (Vmax).

[0005] Therefore, developing a PROD mutant that can retain the high Vmax of wild-type pyranose oxidase while significantly shielding 1,5-AG interference is of great significance for developing high-precision, interference-resistant next-generation blood glucose monitoring devices. Summary of the Invention

[0006] The purpose of this invention is to provide a pyranose oxidase mutant and its application in glucose detection, so as to achieve high specificity and high sensitivity detection of glucose.

[0007] Therefore, the present invention provides the following technical solution.

[0008] The first aspect of the present invention provides a pyranose oxidase mutant obtained by mutating position 155 of the amino acid sequence shown in SEQ ID NO. 2.

[0009] In a preferred embodiment of the present invention, the mutant is one in which glutamine at position 155 is mutated to threonine, alanine, or glycine.

[0010] In a preferred embodiment of the present invention, the mutant is a mutation of glutamine at position 155 of the amino acid sequence shown in SEQ ID NO. 1 to threonine.

[0011] A second aspect of the present invention provides a nucleic acid molecule that encodes a pyranose oxidase mutant as described above.

[0012] In a preferred embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO. 4, or the bases at positions 463-465 are GGT, or the bases at positions 463-465 are GCA.

[0013] A third aspect of the invention provides an expression cassette comprising nucleic acid molecules as described above.

[0014] A fourth aspect of the present invention provides a recombinant vector comprising the nucleic acid molecules as described above.

[0015] In a preferred embodiment of the present invention, the expression vector of the recombinant vector includes the pET28a vector.

[0016] A fifth aspect of the invention provides a host cell that expresses a mutant as described above, contains a nucleic acid molecule as described above, an expression cassette as described above, or is transformed with a recombinant plasmid as described above.

[0017] In a preferred embodiment of the present invention, the host cell is selected from Escherichia coli, yeast, or filamentous fungi.

[0018] A sixth aspect of the present invention provides a method for preparing the pyranose oxidase mutant as described above, comprising: constructing an expression vector containing the mutant gene, transforming it into a host cell, inducing expression, and isolating and purifying the mutant.

[0019] A seventh aspect of the present invention provides the use of the pyranose oxidase mutant as described above in the preparation of glucose detection reagents, biosensors or diagnostic kits.

[0020] In a preferred embodiment of the present invention, the glucose detection reagent, biosensor, or diagnostic kit is used to detect the glucose concentration in a test sample containing 1,5-AG interfering substances.

[0021] In a preferred embodiment of the present invention, the sample to be tested includes, but is not limited to, saliva, tears, interstitial fluid or serum.

[0022] An eighth aspect of the present invention provides a method for detecting glucose concentration in a sample, comprising contacting the sample to be tested with a pyranose oxidase mutant as described above to carry out an oxidation reaction, and detecting the oxidation reaction products or electron transfer signals.

[0023] In a preferred embodiment of the present invention, the oxidation reaction system comprises: 5–15 µL of the sample to be tested, 80–90 µL of the reaction solution, and 5–10 µL of 0.002 mg / mL enzyme working solution.

[0024] In a preferred embodiment of the present invention, the oxidation reaction system comprises: 10 µL of the sample to be tested, 90 µL of the reaction solution, and 10 µL of 0.002 mg / mL enzyme working solution.

[0025] In a preferred embodiment of the present invention, the oxidation reaction system is composed of 0.5 / 10 to 1 / 10 of the test sample, 8 / 10 to 9 / 10 of the reaction solution, and 0.5 / 10 to 1 / 10 of the enzyme working solution, based on a total volume of 10 parts.

[0026] In a preferred embodiment of the present invention, the reaction solution comprises: 0.5-1.0 mM of 4-aminoantipyrrolidone, 0.5-2 U / mL of horseradish peroxidase, and 0.5-2 mM of sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline.

[0027] In a preferred embodiment of the present invention, the reaction solution comprises: 0.75 mM of 4-aminoantipyrrolidone, preferably 1 U / mL of horseradish peroxidase, and 1.5 mM of sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline.

[0028] In a preferred embodiment of the present invention, the detection error of the method in the range of 0.3 mM to 0.6 mM glucose concentration in the sample to be tested is significantly lower than that of the detection error using wild-type pyranose oxidase.

[0029] By employing the above technical solution, the present invention has at least the following advantages: 1. Activity retention: Compared with wild-type pyranose oxidase, the pyranose oxidase mutant Q155T of the present invention retains a similar glucose dose-dependent Vmax, overcoming the defect of other site mutations (e.g., Q155A / G) that lead to a significant decrease in activity.

[0030] 2. Anti-interference ability: In the presence of 0.5 mM 1,5-AG, the pyranose oxidase mutant Q155T of the present invention has significantly better detection accuracy for low concentrations of glucose of 0.3-0.6 mM than the wild type, with an error reduction of 30-50%.

[0031] 3. Extended linear range: The pyranose oxidase mutant Q155T of the present invention extends the linear detection range at the low concentration end to 0.02-5 mM, making it more suitable for the detection of trace amounts of glucose in samples.

[0032] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0033] Figure 1 The diagram shows the recombinant plasmid constructed in this invention; wherein, A is the diagram of the recombinant plasmid pET28a-PROD, and B is the diagram of the recombinant plasmid pET28a-PROD Q155T mutant; Figure 2 The following are protein electrophoresis diagrams of wild-type pyranose oxidase and three pyranose oxidase mutants; Figure 3 The response curves of wild-type pyranose oxidase and three pyranose oxidase mutants to different concentrations of glucose are shown. Figure 4 The response curves of wild-type pyranose oxidase and three pyranose oxidase mutants to different concentrations of 1,5-AG are shown; where A represents 1,5-AG concentrations of 0–100 mM and B represents 1,5-AG concentrations of 0.01–100 mM. Figure 5 The sensitivity tests of wild-type pyranose oxidase and three pyranose oxidase mutants to other monosaccharides are shown. Among them, A is the sensitivity test curve of wild-type PROD to monosaccharides; B is the sensitivity test curve of mutant PROD (Q155T) to monosaccharides; C is the sensitivity test curve of mutant PROD (Q155A) to monosaccharides; and D is the sensitivity test curve of mutant PROD (Q155G) to monosaccharides. Figure 6 Standard curves for wild-type pyranose oxidase and the pyranose oxidase mutant Q155T in artificial saliva are shown; where A is the standard curve for wild-type pyranose oxidase and B is the standard curve for the pyranose oxidase mutant Q155T. Detailed Implementation

[0034] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] This invention discloses an improved pyranose oxidase (PROD) mutant and its applications. The mutant is based on the wild-type sequence from *Trametes ochracea* (a type of *Trametes versicolor*), with a mutation introduced at amino acid position 155 (specifically Q155T). Compared to the wild-type, the mutant of this invention retains a high catalytic rate (Vmax) while significantly reducing its affinity for interfering agents such as 1,5-anhydrous D-glucanol (1,5-AG). The mutant of this invention exhibits good linear response in the glucose concentration range of 0.02–5 mM, and particularly in the presence of physiological concentrations of 1,5-AG interference, it can accurately detect low concentrations (e.g., 0.3–0.6 mM) of glucose, effectively solving the false positive problem caused by interfering agents in existing technologies. This mutant enzyme can be widely used in enzymatic biosensors, non-invasive blood glucose monitoring using saliva and tears, and other fields.

[0036] Specifically, the mutant enzyme constructed in this invention has the following enzymatic properties: ① the maximum reaction rate (Vmax) to glucose is not less than 80% of that of the wild-type enzyme; ② the Michaelis constant (Km) to 1,5-anhydrous D-glucol (1,5-AG) is significantly higher than that of the wild-type enzyme, while Vmax is lower than that of the wild-type enzyme; ③ it exhibits a linear response in the glucose concentration range of 0.02 mM to 5 mM.

[0037] The following detailed description uses specific embodiments.

[0038] The following embodiments involve and mention: 1. Equipment Clean bench (Su Jing An Tai: SW-CJ-2FD, Suzhou An Tai Air Technology Co., Ltd.); Shaking incubator (ZQLY-180E, Shanghai Zhi Chu Instrument Co., Ltd.).

[0039] 2. Reagents Escherichia coli BL21(DE3) chemicompetent cells (Nanjing Novizan Biotechnology Co., Ltd., C504-03); plasmid miniprep kit (Solarbio, D1100); GST-tagged protein purification kit (Beyotime Biotechnology Co., Ltd., P2262); protein concentration assay kit (Beyotime Biotechnology Co., Ltd., P0006); 180 kDa Prestained Protein Marker (Nanjing Novizan Biotechnology Co., Ltd., MP102-01); ampicillin (CAS: 69-52-3, Maclean's, A800429); isopropyl-β-D-thiogalactopyranoside (CAS: 367-93-1, Maclean's, I6148); One-Step PAGE Gel Fast Preparation Kit (8%).

[0040] Example 1: Expression and purification of PROD mutant protein 1. Sequence optimization of PROD and construction of mutants Using the wild-type pyranose oxidase PROD gene from Trametes ochracea (nucleotide sequence as shown in SEQ ID NO. 1, amino acid sequence as shown in SEQ ID NO. 2) as a template, Q155T, Q155A and Q155G mutations were introduced respectively using site-directed mutagenesis.

[0041] SEQ ID NO. 1 SEQ ID NO. 2 MSTSSSDPFFNFAKSSFRSAAAQKASASSLPPLPGPDKKVPGMDIKYDVVVGSGPIGCTYARELVGAGYKVAMFDIGEIDSGLKIGAHKKNTVEYQKNIDKFVNVIQGQLMSVSVPVNTLVVDTLSPTSWQASTFFVRNGSNPEQDPLRNLSGQ AVTRVVGGMSTHWTCATPRFDREQRPLLVKDDADADDAEWDRLYTKAESYFQTGTDQFKESIRHNLVLNKLTEEYKGQRDFQQIPLAATRRSPTFVEWSSANTVFDLQNRPNTDAPEERFNLFPAVACERVVRNALNSEIESLHIHDLISGDRFEI KADVYVLTAGAVHNTQLLVNSGFGQLGRPNPANPPELLPSLGSYITEQSLVFCQTVMSTELIDSVKSDMTIRGTPGELTYSVTYTPGASTNKHPDWWNEKVKNHMMQHQEDPLPIPFEDPEPQVTTLFQPSHPWHTQIHRDAFSYGAVQQSIDSRL IVDWRFFGRTEPKEENKLWFSDKITDAYNMPQPTFDFRFPAGRTSKEAEDMMTDMCVMSAKIGGFLPGSLPQFMEPGLVLHLGGTHRMGFDEKEDNCCVNTDSRVFGFKNLFLGGCGNIPTAYGANPTLTAMSLAIKSCEYIKQNFTPSPFTSEAQ To facilitate the successful expression of the pyranose oxidase gene in *E. coli*, the wild-type pyranose oxidase PROD gene sequence was further optimized. The optimized base sequence is shown in SEQ ID NO. 3. SEQ ID NO. 3 In the above base sequence, the CAG base at positions 463-465 corresponds to the Q155 amino acid. Verification showed that after translating the optimized base sequence into an amino acid sequence, no errors were found when compared with the protein amino acid sequence of WT (wild-type) pyranose oxidase.

[0042] Therefore, the pyranose oxidase mutant Q155T is formed by changing the base CAG to ACC at positions 463-465 of the wild-type pyranose oxidase, that is, completing the mutation from amino acid Q to T at position 155. The nucleotide sequence of the resulting pyranose oxidase mutant Q155T is shown in SEQ ID NO. 4: SEQ ID NO. 4 Construction of pyranose oxidase mutants Q155A and Q155G: Based on the same principle as above, mutating the CAG base at positions 463-465 of wild-type pyranose oxidase to GGT results in pyranose oxidase mutant Q155G; mutating the CAG base at positions 463-465 of wild-type pyranose oxidase to GCA results in pyranose oxidase mutant Q155A.

[0043] 2. Constructing a reorganization vehicle The target gene was synthesized according to standard procedures. Using the *E. coli* expression vector plasmid pET28a as the expression vector, the wild-type pyranose oxidase gene (nucleotide sequence as shown in SEQ ID NO. 3) or the pyranose oxidase mutant Q155T gene (nucleotide sequence as shown in SEQ ID NO. 4) was inserted to construct recombinant plasmids pET28a-PROD and pET28a-PROD(Q155T). The plasmid maps are shown below. Figure 1 As shown.

[0044] The insertion sites of the other two pyranose oxidase mutants, Q155A and Q155G, are the same as those of PROD Q155T, and will not be repeated here. The corresponding recombinant plasmids pET28a-PROD(Q155A) and pET28a-PROD(Q155G) are obtained.

[0045] 3. Protein expression and purification 3.1 Transformation into expression bacteria In a clean bench, 2 µL of recombinant plasmid pET28a-PROD (or mutant recombinant plasmids pET28a-PROD MB(Q155T), pET28a-PROD(Q155A), or pET28a-PROD(Q155G)) was added to 50 µL of *E. coli* BL21(DE 3) competent cells and mixed. The mixture was then incubated on ice for 30 min. Heat shock: The mixture was placed in a water bath at 42°C for 90 s and then immediately removed. After incubating on ice for 3 min, 900 µL of antibiotic-free liquid LB medium was added to the mixture and the mixture was placed in a shaker at 37°C and shaken at 220 rpm for 45 min. Finally, the mixture was centrifuged at 2000 rpm for 2 min. Discard most of the supernatant in a clean bench, leaving about 50µL of culture medium to resuspend the bacteria. Aspirate and add the bacteria to a solid LB culture plate containing kanamycin (Kan) antibiotic (1 mg / mL). Spread the bacterial solution evenly using the bead coating method, and invert the plate to incubate overnight at 37°C.

[0046] 3.2 Induced Expression Perform the procedure in a clean bench. Select a single colony from the culture plate and add 5 mL of liquid LB medium containing 1 mg / mL of Kansin antibiotic. Incubate overnight on a shaker at 37°C and 220 rpm. Then, in the same clean bench, transfer the overnight culture to eight Erlenmeyer flasks containing 200 mL of liquid LB medium at a 1:100 ratio. Add Kansin antibiotic and incubate at 37°C and 220 rpm on a shaker. After approximately 3–4 hours of incubation, take 1 mL of the culture and measure the OD (octane rating). 600 Value, until OD 600 Between 0.6 and 0.8, add IPTG (100 mM) as an inducer to a final concentration of 0.1 mM into an Erlenmeyer flask for induction expression. Incubate at 16°C and 220 rpm for approximately 16 hours. After induction, centrifuge at 4°C and 12000 rpm for 20 minutes, discard the supernatant, and collect the bacterial culture. Wash the bacterial culture once with PBS, then centrifuge at 4°C and 12000 rpm for 20 minutes, discarding the supernatant.

[0047] 3.3 Purification of HIS-tagged fusion proteins 3.3.1 Buffer Preparation Lysis equilibration buffer (LE Buffer): 50 mM NaH2PO4, 300 mM NaCl, adjusted to pH 7.4; Washing buffer (WB Buffer): 50 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, adjusted to pH 7.4; Elution buffer (EB Buffer): 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, adjusted to pH 7.4.

[0048] Note: The prepared buffer solution needs to be filtered through a 0.45μm filter membrane to prevent the purification column from clogging.

[0049] 3.3.2 Sample Preparation Resuspend the collected bacterial cells in an appropriate amount of LE Buffer (a protease inhibitor may be added), and sonicate to disrupt the cells: operate for 3 seconds, cool for 4 seconds, and continue for 30–45 minutes. Then centrifuge at 4°C and 12,000 rpm for 20 minutes, collect the supernatant, and filter through a 0.45 μm filter membrane.

[0050] The purification column was packed with nickel affinity and equilibrated with LE Buffer in preparation for sample loading.

[0051] 3.3.3 Purification by Chromatography The column was slowly eluted with WB buffer to remove contaminating proteins, and the removal of contaminating proteins was monitored by passing the eluent. After the contaminating proteins were completely removed, the target protein was eluted with EB buffer and collected until the target protein was completely eluted. The column was then washed again with EB buffer, and finally equilibrated with LE buffer and stored in 20% ethanol.

[0052] 3.4 SDS-PAGE electrophoresis Prepare electrophoresis gels according to the One-Step PAGE Gel Fast Preparation Kit (8%). Protein sample preparation: Take 50 µL of protein sample, add 10 µL of Protein Loading Buffer, mix well, boil in boiling water for 10 min, and let cool and stand. After installing the electrophoresis gel, remove the comb, add the protein marker and sample sequentially, and turn on the power switch to a constant voltage of 120V for electrophoresis. After approximately 2 hours of electrophoresis, when the protein marker has dispersed to a suitable state, turn off the power, remove the electrophoresis gel, and stain with Coomassie Brilliant Blue rapid staining solution. Clear bands will be visible after 30 min; photograph and record. Electrophoresis images of wild-type PROD and pyranose oxidase mutants Q155A, Q155G, and Q155T are shown below. Figure 2 .

[0053] like Figure 2 The electrophoresis diagrams of PROD shown correspond from left to right to the protein electrophoresis diagrams of wild-type PROD, mutant Q155A, mutant Q155T, and mutant Q155G, respectively. Standard proteins of different sizes are also shown and labeled at 70 kd.

[0054] Example 2: Enzyme kinetics analysis of pyranose oxidase mutants The activity of the obtained pyranose oxidase mutants was evaluated using enzyme-linked spectrometry (measuring hydrogen peroxide production at 550 nm) to verify the substrate selectivity of different PROD mutants. The specific steps included: 1. Preparation of buffer solution and reaction solution Buffer solution: 200 mM HEPES buffer solution (purchased from Macklin), adjusted to pH 7.6.

[0055] Reaction solution: The reaction solution prepared based on this buffer contains: 0.75 mM 4-aminoantipyrrolidone (4-AAP) (Macklin), 1 U / mL horseradish peroxidase (HRP) (Macklin), and 1.5 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS) (Macklin).

[0056] Enzyme working solutions: Pyranose oxidase (PROD): 0.002 mg / mL (Seebio); Pyranose oxidase mutant Q155T: 0.002 mg / mL, Pyranose oxidase mutant Q155A: 0.012 mg / mL and Pyranose oxidase mutant Q155G: 0.007 mg / mL.

[0057] Preparation of different sugar concentrations in the enzyme sensitivity assay: Sugars included glucose, 1,5-AG, D-mannoheptulose, fructose, galactose, D-ribose, and L-arabinose (all sugars were purchased from Macklin). The solvent for the sugar solutions was pure water, and the concentrations (mM) included: 0, 0.097656, 0.195313, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100.

[0058] Preparation of glucose solutions of different concentrations for low-concentration glucose detection: The solvent for glucose solutions is pure water, and the concentrations (mM) include: 0, 0.004, 0.009, 0.019, 0.039, 0.078, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5.

[0059] 2. Specific operating procedures and equipment The reaction system has a capacity of 100 µL: 10 µL of sugar solutions of different concentrations (ranging from 0 to 5 mM and 0 to 100 mM) + 80 µL of reaction solution + 10 µL of enzyme working solution.

[0060] The above solutions were added to 96-well plates and mixed thoroughly with a shaker (Mini shaker MH-1, Kulin-Bell Lab Instruments). The plates were then placed in an incubator (Shanghai Xinmiao) and incubated at 37°C for 15 minutes. The absorbance OD value was then measured using a microplate reader (CLARIOstar) at a wavelength of 550 nm.

[0061] 3. Results and Analysis 3.1 Sensitivity to glucose like Figure 3 As shown, the dose-dependent curves of glucose for the pyranose oxidase mutant Q155T indicate that the Vmax per unit protein concentration is similar to that of the wild type (wild-type Vmax is approximately 30.28 µmol / mg protein / 15 min, while Q155T is approximately 28.79 µmol / mg protein / 15 min, a difference of approximately 5%). In contrast, the Vmax of the pyranose oxidase mutants Q155A and Q155G is significantly reduced (a decrease of >70%, to 7.47 µmol / mg protein / 15 min and 4.66 µmol / mg protein / 15 min, respectively).

[0062] 3.2 Sensitivity to 1,5-AG like Figure 4 As shown in Figure A, compared with wild-type pyranose oxidase, the mutant pyranose oxidase Q155 exhibits a significantly reduced Vmax value for 1,5-AG. The Vmax value for wild-type pyranose oxidase is 62.36 µmol / mg protein / 15 min, while the Vmax value for pyranose oxidase Q155T is 25.63 µmol / mg protein / 15 min, a reduction of approximately 60%, while the Vmax value for pyranose oxidase Q155T is 79.43 mM, compared to 45.55 mM for wild-type pyranose oxidase. Regarding the Vmax / Km ratio, the ratio for wild-type pyranose oxidase is 1.37, while that for pyranose oxidase Q155T is 0.32, a reduction of 76%. This suggests that compared with wild-type PROD, the mutant PROD (Q155T) has severely impaired sensitivity to 1,5-AG. Figure 4 B more clearly demonstrates the sensitivity of pyranose oxidase Q155T to 1,5-AG; the catalytic reaction of pyranose oxidase Q155T only begins when the concentration of 1,5-AG exceeds approximately 10 mM.

[0063] 3.3 Sensitivity of PROD mutants to other monosaccharides (such as galactose and xylose) like Figure 5As shown, to facilitate comparison of the sensitivity of wild-type oxidase and the three pyranose oxidase mutants to other monosaccharides, the maximum glucose response of each enzyme was taken as 100%, and the response curves for other monosaccharides are relative to their glucose responses (i.e., percentages of the maximum glucose response). It can be seen that wild-type PROD exhibits excellent catalytic responses to glucose and 1,5-AG, as well as galactose. However, pyranose oxidases Q155T, Q155A, and Q155G all show decreased sensitivity to 1,5-AG and complete loss of sensitivity to other monosaccharides (including D-heptose, fructose, galactose, ribose, and arabinose).

[0064] Example 3: Interference resistance detection under simulated body fluid environment The pyranose oxidase mutant Q155T was used to detect low glucose levels in artificial saliva samples. While adding different concentrations of glucose, 0.5 mM 1,5-AG was added as an interfering agent to test the anti-interference ability of the pyranose oxidase mutant Q155T in glucose detection. The specific steps included: 1. Preparation of buffer solution and reaction solution Buffer solution: 200 mM HEPES buffer solution (purchased from Macklin), adjusted to pH 7.6.

[0065] Reaction solution: The reaction solution prepared based on this buffer contains: 0.75 mM 4-aminoantipyrrolidone (4-AAP) (Macklin), 1 U / mL horseradish peroxidase (HRP) (Macklin), and 1.5 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS) (Macklin).

[0066] Enzyme working solution: Pyranose oxidase (PROD) (Seebio): 0.25 U / mL pyranose oxidase; Pyranose oxidase mutant Q155T: 0.002 mg / mL.

[0067] Preparation of glucose solutions of different concentrations: The solvents for glucose solutions are pure water and artificial saliva (formulas are shown in Table 1). The solvents are divided into two groups: (1) pure water or artificial saliva group; (2) pure water or artificial saliva group containing 0.5 mM 1,5-AG; the glucose concentration range is (0-5 mM), including 0, 0.004, 0.009, 0.019, 0.039, 0.078, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 (mM).

[0068] Table 1. Preparation of artificial saliva (all compounds were purchased from Macklin) ; 2. Specific operating procedures and equipment: The reaction system has a capacity of 100 μL: 10 μL glucose solution (0-5 mM) + 80 μL reaction solution + 10 μL enzyme working solution; The above solutions were added to 96-well plates and mixed thoroughly with a shaker (Mini shaker MH-1, Kulin-Bell Lab Instruments). The plates were then placed in an incubator (Shanghai Xinmiao) and incubated at 37°C for 15 minutes. The absorbance OD value was then measured using a microplate reader (CLARIOstar) at a wavelength of 550 nm.

[0069] 3. Results Analysis and Analysis 3.1 Detection range of wild-type PROD and mutant (Q155T) in glucose detection like Figure 6 As shown in A, the linear response of wild-type PROD to glucose detection ranged from 0.039 to 2.5 mM. Figure 6 B shows that the linear range of the pyranose oxidase mutant Q155T in response to glucose is 0.019–5 mM.

[0070] 3.2 Interference effect of 1,5-AG on low glucose concentration monitoring 0.5 mM 1,5-AG was added to glucose solutions dissolved in artificial saliva in the range of 0.3–2.5 mM, and the results were detected using Q155T and wild-type PROD, respectively. The results are shown in Table 2.

[0071] Table 2 Comparison of glucose recovery rates in the presence of 0.5 mM 1,5-AG interference ; As shown in Table 2, at glucose concentrations of 0.3125 mM and 0.625 mM, the glucose concentration monitoring results using wild-type PROD were approximately 30-50% higher than the theoretical values. For example, the measured value for an actual glucose concentration of 0.3125 mM was 0.418 ± 0.007 mM, which was 34 ± 2% higher than the actual value. However, the glucose concentration detection using the mutant PROD (Q155T) was unaffected (error < 5%), for example, the measured value for an actual glucose concentration of 0.3125 mM was 0.3076 ± 0.03 mM. When the glucose concentration increased to 1.2 mM and 2.5 mM, neither was affected by 1,5-AG.

[0072] The above results demonstrate the superiority of the mutant PROD (Q155T) in detecting very low glucose concentrations, and it can be further optimized into a commercial diagnostic product, especially for the detection of low glucose concentrations.

[0073] 3.3 Data Analysis Conclusions 3.3.1 Low concentration range (0.3–0.6 mM) This is the critical range for glucose detection in saliva / tears. The wild-type enzyme exhibited severe signal overestimation (134%–150%), clearly demonstrating its inability to distinguish low concentrations of glucose from background 1,5-AG. In contrast, the Q155T mutant showed detection values ​​close to the theoretical values ​​(98%–105%), with a p-value indicating statistical significance. This demonstrates that Q155T successfully eliminated the 30–50% detection error caused by 1,5-AG.

[0074] 3.3.2 High concentration range (> 1.25 mM) As glucose concentration increases, competitive advantage reduces the impact of interference, and the two tend to behave similarly.

[0075] The above results confirm that the mutant Q155T of the present invention is particularly suitable for high-precision detection of low-concentration glucose, solving the pain points of existing commercial enzymes in non-invasive blood glucose monitoring.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A pyranose oxidase mutant, characterized in that, The mutant was obtained by mutating position 155 of the amino acid sequence shown in SEQ ID NO. 2; The mutant is one in which glutamine at position 155 is mutated to threonine, alanine, or glycine.

2. The pyranose oxidase mutant according to claim 1, characterized in that, The mutant is formed by mutating glutamine at position 155 of the amino acid sequence shown in SEQ ID NO. 1 to threonine.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the pyranose oxidase mutant of claim 1; The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 4, or the bases at positions 463-465 are GGT, or the bases at positions 463-465 are GCA.

4. An expression box, characterized in that, The expression cassette comprises the nucleic acid molecule of claim 3.

5. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule of claim 3; Preferably, the expression vector of the recombinant vector includes the pET28a vector.

6. Expressing a host cell having the mutant of claim 1 or 2, containing the nucleic acid molecule of claim 3, the expression cassette of claim 4, or transformed with the recombinant plasmid of claim 5; Preferably, the host cell is selected from Escherichia coli, yeast, or filamentous fungi.

7. A method for preparing the pyranose oxidase mutant according to claim 1 or 2, characterized in that, include: An expression vector containing the mutant gene was constructed, transformed into a host cell, induced to express, and then isolated and purified to obtain the mutant.

8. The application of the pyranose oxidase mutant according to claim 1 in the preparation of glucose detection reagents, biosensors or diagnostic kits.

9. The application according to claim 8, characterized in that, The glucose detection reagent, biosensor, or diagnostic kit is used to detect the glucose concentration in a test sample containing 1,5-AG interfering substances. The samples to be tested include, but are not limited to, saliva, tears, interstitial fluid, or serum.

10. A method for detecting glucose concentration in a sample, characterized in that, This includes contacting the sample to be tested with the pyranose oxidase mutant of claim 1 or 2 to carry out an oxidation reaction, and detecting the oxidation reaction products or electron transfer signals; The method exhibits a significantly lower detection error in the 0.3 mM–0.6 mM glucose concentration range of the test sample compared to the detection error using wild-type pyranose oxidase.

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