Glucose dehydrogenase, encoding gene thereof, recombinant carrier, genetically engineered bacteria and application

Abstract

The application discloses glucose dehydrogenase, a coding gene thereof, a recombinant carrier, genetically engineered bacteria and application, relates to the field of molecular biology and enzyme engineering, aims to overcome the defects of insufficient heat stability and poor specificity of prior art, and specifically provides glucose dehydrogenase with SEQ ID NO:1 or SEQ ID NO:2, a coding gene, a recombinant carrier, a recombinant engineering strain and a method for preparing glucose dehydrogenase, and also provides application in blood glucose detection test paper and blood glucose sensors. A strain of aspergillus niger for producing glucose dehydrogenase is also provided, which was preserved in the China General Microbiological Culture Collection Center on May 29, 2026, and the preservation number is CGMCC No. 42719. The application still maintains a relatively high residual enzyme activity after high-temperature treatment, is not interfered by dissolved oxygen in a sample, does not need to add exogenous coenzyme, and has high specificity for glucose.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and enzyme engineering. More specifically, this invention relates to a glucose dehydrogenase, its encoding gene, recombinant vector, genetically engineered bacteria, and its applications. Background Technology

[0002] Accurate monitoring of blood glucose concentration is crucial for the diagnosis, treatment, and long-term management of diabetes. Currently, enzymes used for blood glucose detection mainly fall into two categories: glucose oxidase (GOX) and glucose dehydrogenase (GDH). GOX was the first enzyme used in blood glucose detection. When it catalyzes the oxidation of glucose, it uses oxygen molecules as the natural electron acceptor. The hydrogen peroxide produced during the reaction needs to be detected by electrodes. This mechanism makes the measurement results susceptible to fluctuations in the dissolved oxygen concentration in the sample, especially under hypoxic or oxygen-enriched conditions, which significantly limits its detection accuracy.

[0003] To address the problem of oxygen interference, researchers developed PQQ-GDH, a coenzyme using pyrroloquinoline quinone (PQQ). This enzyme is oxygen-independent and requires no exogenous NAD(P)+ cofactor, and was once considered the most promising alternative enzyme. However, PQQ-GDH has inherent drawbacks: firstly, the binding force between the PQQ coenzyme and the enzyme protein is weak, leading to easy dissociation and activity decay during storage or use; secondly, the enzyme has insufficient selectivity for glucose and exhibits significant cross-reactivity with interfering sugars such as maltose and xylose, which has caused false hyperglycemia due to interference from infusion components in clinical practice, resulting in serious medical accidents. Furthermore, PQQ-GDH is mostly derived from cell membrane components, making extraction and purification difficult and hindering industrial production. In addition, FAD-GDH, using flavin adenine dinucleotide (FAD) as a coenzyme, has also attracted attention. This type of enzyme combines the advantages of being oxygen-independent and requiring no exogenous coenzyme, and its specificity for glucose is superior to PQQ-GDH. However, the current production level of FAD-GDH is still insufficient to meet the needs of large-scale industrialization, and FAD-GDH from certain sources still exhibits a certain degree of cross-reactivity with xylose, which may affect the reliability of results in specific clinical testing scenarios.

[0004] Therefore, there is an urgent need to develop a technical solution that can overcome the above-mentioned defects. Summary of the Invention

[0005] One objective of this invention is to provide a glucose dehydrogenase, its encoding gene, recombinant vector, genetically engineered bacteria, and applications, in order to overcome the shortcomings of existing glucose dehydrogenases, such as insufficient thermostability and poor specificity.

[0006] In order to achieve these objects and other advantages of the present invention, according to one aspect of the present invention, a glucose dehydrogenase having the amino acid sequence of SEQ ID NO: 1 is provided.

[0007] The present invention provides a glucose dehydrogenase having the amino acid sequence of SEQ ID NO: 2.

[0008] The present invention also provides a gene encoding the glucose dehydrogenase described herein.

[0009] The present invention also provides a recombinant vector comprising the said gene.

[0010] The present invention also provides recombinant engineered strains containing the recombinant vector.

[0011] The present invention also provides a method for preparing glucose dehydrogenase, comprising: S1: culturing the recombinant engineered strain; S2: recovering the glucose dehydrogenase from the recombinant engineered strain or a culture of the recombinant engineered strain.

[0012] The present invention also provides a *Aspergillus niger* strain for producing the glucose dehydrogenase, the *Aspergillus niger* strain being classified as *Aspergillus niger* (…). Aspergillusniger The specimen was deposited on May 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42719. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0013] The present invention also provides the application of the glucose dehydrogenase in the preparation of blood glucose test strips.

[0014] The present invention also provides the application of the glucose dehydrogenase in the preparation of a blood glucose sensor.

[0015] The present invention has at least the following beneficial effects: This invention provides a glucose dehydrogenase that maintains high residual enzyme activity even after high-temperature treatment, exhibiting significantly superior thermal stability compared to existing technologies. This effectively solves the problem of insufficient stability caused by heat inactivation during storage and use of existing enzymes. The glucose dehydrogenase of this invention uses FAD as a coenzyme, and its catalytic glucose oxidation reaction is unaffected by dissolved oxygen in the sample, overcoming the inherent defect of traditional glucose oxidases that leads to biased detection results due to oxygen dependence. It eliminates the need for exogenous coenzymes such as NAD(P)+ or PQQ, avoiding the activity decay problem caused by PQQ-GDH coenzyme dissociation, and significantly improving the stability and ease of operation of the detection system. The glucose dehydrogenase of this invention exhibits high specificity for glucose and low cross-reactivity with various interfering sugars such as maltose, xylose, galactose, and fructose. This effectively avoids false increases in blood glucose levels caused by interfering substances in clinical testing, greatly improving the accuracy and safety of detection results, and making it suitable for clinical diagnostic scenarios with strict accuracy requirements.

[0016] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0017] Figure 1 The figure shows the pH-enzyme activity curves; in the figure, GDH-AF is the control enzyme, GDH represents the glucose dehydrogenase corresponding to SEQ ID NO: 1, and GDH-mut represents the glucose dehydrogenase corresponding to SEQ ID NO: 2.

[0018] Figure 2 The figures show enzyme activity curves after 17 h at different pH values. In the figure, GDH-AF is the control enzyme, GDH represents the glucose dehydrogenase corresponding to SEQ ID NO:1, and GDH-mut represents the glucose dehydrogenase corresponding to SEQ ID NO:2.

[0019] Figure 3 The figure shows the temperature-enzyme activity curves; in the figure, GDH-AF is the control enzyme, GDH represents the glucose dehydrogenase corresponding to SEQ ID NO: 1, and GDH-mut represents the glucose dehydrogenase corresponding to SEQ ID NO: 2.

[0020] Figure 4 The figures show enzyme activity curves after being kept at different temperatures for 20 min. In the figure, GDH-AF is the control enzyme, GDH represents the glucose dehydrogenase corresponding to SEQ ID NO: 1, and GDH-mut represents the glucose dehydrogenase corresponding to SEQ ID NO: 2.

[0021] The strain of Aspergillus niger is classified as Aspergillus niger (Aspergillus niger Aspergillus niger The specimen was deposited on May 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42719. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0022] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0023] It should be understood that terms such as "having," "comprising," and "including" used in the embodiments of this application do not exclude the presence or addition of one or more other elements or combinations thereof. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are only used to explain the relative positional relationship and movement of components in a specific posture. If the specific posture changes, the directional indication will change accordingly. When an element is referred to as "fixed to" or "set on" another element, it can be directly on the other element or may have an intervening element present. When an element is referred to as "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element through an intervening element. Descriptions involving "first," "second," etc., in the embodiments of this application are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of those features.

[0024] It should be noted that the technical solutions of the various embodiments of this application can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this application.

[0025] An embodiment of this application provides a glucose dehydrogenase having the amino acid sequence shown in SEQ ID NO: 1 (also referred to as GDH in this part) or SEQ ID NO: 2 (also referred to as GDH-mut in this part).

[0026] The sequence of SEQ ID NO: 1 is: KQTTTYDYIVVGGGTSGLVVANRLSENPDVSVLLLEAGASVFNNPDVTNANGYGLAFGSAIDWQYQSIQQSYAGGKQQVLRAGKALGGTSTINGMAYTRAKDVQIDVWQKLGNEGWTWKDLLPYYLKSEQLTAPTSSQVPAGAAYNPACNGKEGPLKVGWSGSLASGNLSVALARTFQAAGVPWVEDVNCGKMRGFNIYPSTLDVDLNVREDAARAYYFPYDDRKNLHLLENTTANRLFWKQGSAEEAIADGVEISSADGKVTRVHAKKEVIISAGALRSPLILELPGVGNPTILKKNNITPRVDLPTVGENLQDQFNQGMAGEGYGVLAGASTVTYPSISDVFPQETDSIVASLISQLSDYAAATVKVSQGHMKQEDLERLYQLQFDLIVKDKVPIAEILFHPGGGNAVSSEFWGLLPFARGNIYISSNDPTAPAAINPNYFMFEWDGKSQAGIAKYIRKILRSAPLNKLIAKETKPGLSEIPATAADEKWVEWLKANYRSNFHPVGTAAMMPRSIGGVVDNRLRVYGTSQVRVVDASVLPFQVCGHLVSTLYAVAERASDLIKEDAKSA The sequence of SEQ ID NO: 2 is as follows: KQTTTYDYIVVGGGTSGLVVANRLSENPDVSVLLLEAGASVFNNPDVTNANGYGLAFGSAIDWQYQSIQQSYAGGKQQVLRAGKALGGTSTINGMAYTRAKDVQIDVWQKLGNEGWTWKDLLPYYLKSEQLTAPTSSQVPAG AAYNPACNGKEGPLKVGWSGSLASGNLSVALARTFQAAGVPWVEDVNCGKFRGFNIYPSTLDVDLNVREDAARAYYFPYDDRKNLHLLENTTANRLFWKQGSAEEAIADGVEISSADGKVTRVHAKKEVIISAGALRSPLILE LPGVGNPTILKKNNITPRVDLPTVGENLQDQFNQGMAGEGYGVLAGASTVTYPSISDVFPQETDSIVASLISQLSDYAAPTVKVSQGHMKQEDLERLYQLQFDLIVKDKVPIAEILFHPQGGNAVSSEFWGLLPFARGNIYIS SNDPTAPAAINPNYFMFEWDGKSQAGIAKYIRKILRSAPLNKLIAKETKPGLSEIPATAADEKWVEWLKANYRSNFHPVGTAAMMPRSIGGVVDNRLRVYGTSQVRVVDASVLPFQVCGHLVSTLYAVAERASDLIKEDAKSA It should be noted that the glucose dehydrogenase sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 above do not contain a histidine tag. During recombinant expression, a histidine tag coding sequence can be fused to the 5' or 3' end of the target gene in the expression vector, depending on purification requirements, to facilitate rapid purification via nickel column affinity chromatography.

[0027] Those skilled in the art should understand that, without substantially affecting glucose dehydrogenase activity, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions can be made to the sequence. According to hydrophilicity / hydrophobicity classification, hydrophobic amino acids include A, C, I, L, M, F, W, and V; neutral amino acids include G, H, P, S, T, and Y; and hydrophilic amino acids include R, N, D, Q, E, and K. According to charge classification, positively charged amino acids are R, H, and K; negatively charged amino acids are D and E; and uncharged amino acids are A, N, C, Q, G, I, L, M, F, P, S, T, W, Y, and V. According to chemical properties classification, aliphatic amino acids include A, G, I, L, P, and V; aromatic amino acids include F, W, and Y; sulfur-containing amino acids include C and M; hydroxyl-containing amino acids are S and T; basic amino acids are R, H, and K; acidic amino acids are D and E; and amide amino acids are N and Q. Substitutions between amino acid residues of the same class (such as K replacing R, L replacing I, D replacing E, S replacing T, A replacing G, Q replacing N, P replacing G, F replacing W, A replacing V, C replacing M, etc.) generally do not change the protein's stereostructure and functional activity. SEQ ID NO: 2 is obtained by mutating amino acid M to F, amino acid A to P, and amino acid G to Q in SEQ ID NO: 1.

[0028] Embodiments of this application also provide a polynucleotide sequence encoding the glucose dehydrogenase, specifically the gene encoding SEQ ID NO: 1 or SEQ ID NO: 2, as shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0029] This application also provides a recombinant vector containing the gene encoding glucose dehydrogenase. The vector can be any nucleic acid molecule capable of self-replication in a host cell or integration into the host chromosome, such as plasmids, bacteriophages, granules, or viral vectors. In a preferred embodiment, the vector is the Pichia pastoris expression vector pPIC9K, which carries a strong promoter of alcohol oxidase 1 (AOX1) and a secretion signal peptide sequence, enabling efficient transcription and translation of the target gene and secretion of the recombinant protein into the extracellular culture medium, greatly simplifying subsequent purification steps. The construction method of the recombinant vector includes: double digestion of the codon-optimized glucose dehydrogenase gene with restriction endonucleases (such as EcoRI and SalI), ligation with the similarly digested pPIC9K vector backbone using T4 DNA ligase, transformation into *E. coli* DH5α, and screening for positive clones. Besides pPIC9K, other suitable expression vectors, such as pPICZα and pGAPZα, and expression systems suitable for mammalian cells, insect cells, fungi, or prokaryotes can also be used in this application. The protection scope of the vector is not limited to a specific plasmid, but covers any nucleic acid construct that can carry and express the glucose dehydrogenase gene of this application, including all forms containing regulatory elements such as selection markers, origin of replication, promoters, terminators, and signal peptides.

[0030] The embodiments of this application also provide recombinant engineered strains containing the recombinant vector, i.e., genetically engineered microorganisms capable of expressing glucose dehydrogenase obtained by introducing the recombinant vector into host cells through transformation or transfection. The host cells can be prokaryotic cells (such as *Escherichia coli*) or eukaryotic cells (such as yeast, *Aspergillus niger*, *Aspergillus oryzae*, *Aspergillus nidus*, insect or mammalian cells), but *Pichia pastoris* strain X-33 or *Aspergillus niger* strain are preferred because these strains have a methanol-inducible AOX1 promoter, suitable for high-level secretory expression of exogenous proteins, and yeast itself secretes fewer contaminating proteins, which is beneficial for subsequent purification; *Aspergillus niger* strain is suitable for expressing glucose dehydrogenase more closely to its native state, and the fermentation broth contains fewer contaminating proteins, making it suitable for industrial production. Specifically, the linearized recombinant vector pPIC9K-GDH or pPIC9K-GDH-mut is introduced into X-33 competent cells using electroporation, and positive transformants are obtained through G418 resistance selection and PCR identification. Recombinant engineered strains include not only strains containing free vectors but also strains whose vectors are stably inherited after integration into the host chromosome. Furthermore, the scope of protection covers any host cell with glucose dehydrogenase expression capability obtained by transformation of the recombinant vector, regardless of its specific genetic background or culture method, as long as it contains the gene of this application and can produce an enzyme protein with the corresponding amino acid sequence, it falls within the scope of protection of this application.

[0031] This application also provides a method for preparing glucose dehydrogenase, comprising: S1: culturing the recombinant engineered strain in a suitable culture medium to express the glucose dehydrogenase gene; S2: recovering the glucose dehydrogenase protein from the cells of the recombinant engineered strain or from the culture of the strain (such as fermentation supernatant). Specifically, the culture step can employ batch fermentation or fed-batch fermentation. For the Pichia pastoris recombinant strain, it is first cultured in BMGY medium (containing 1% glycerol) to the logarithmic phase, then replaced with BMMY medium and 0.5%~3% methanol is added for induction. The induction temperature is usually controlled at 25-30 °C, the pH is maintained at 5.0-8.0, and the induction time is approximately 72-96 hours. During fermentation, the induction concentration is maintained by adding methanol, and the feeding rate is controlled by dissolved oxygen feedback. The glucose dehydrogenase of this application belongs to the flavin adenine dinucleotide (FAD) dependent enzyme. FAD is the prosthetic group necessary for its catalytic activity, which is tightly bound to the active site of the enzyme protein by non-covalent bonds. In recombinant expression systems, FAD is not an exogenous additive but is synthesized endogenously by the host cell (such as Pichia pastoris) through its own metabolic pathways. During growth, the host cell utilizes the riboflavin (vitamin B2) biosynthesis pathway, phosphorylating and adenyling riboflavin to generate FAD under the sequential catalysis of riboflavin kinase and FAD synthase. The newly synthesized FAD, with the assistance of molecular chaperones, spontaneously assembles into the folded structure of the glucose dehydrogenase polypeptide chain expressed in recombinant expression, forming a holoenzyme with complete catalytic function. To ensure an adequate supply of FAD to the host cell, riboflavin or yeast extract (naturally containing riboflavin and FAD precursors) can be added to the fermentation medium to promote the full formation of the holoenzyme and increase enzyme activity per unit. In enzyme-catalyzed reactions, the mechanism of action of FAD is as follows: When a glucose molecule enters the enzyme's active site, glucose is oxidized to D-gluconic acid-δ-lactone, while FAD, acting as an electron acceptor, is reduced to FADH2. Subsequently, the reduced FADH2 transfers electrons to electron mediators in the reaction system (such as PMS, potassium ferricyanide, etc.) and is re-oxidized back to FAD, completing one catalytic cycle. This electron transport chain does not involve oxygen molecules, therefore the glucose dehydrogenase of this application is not affected by dissolved oxygen in detection applications. Furthermore, the FAD cofactor is firmly bound to the enzyme protein and is not easily dissociated under normal storage and use conditions. Therefore, there is no need to add coenzymes such as FAD or NAD(P)+ to the detection system, significantly simplifying the operation process and reducing detection costs.

[0032] The recovery steps include: centrifugation or filtration to remove bacterial cells, collecting the supernatant containing secretory glucose dehydrogenase; then, a combination of purification techniques such as ammonium sulfate precipitation, ion exchange chromatography (e.g., Q-Sepharose), affinity chromatography (e.g., Ni-NTA column, using a C-terminal His tag), and gel filtration chromatography (e.g., Superdex 200) are used to finally obtain high-purity glucose dehydrogenase protein. During the purification process, the FAD cofactor remains bound to the enzyme protein. The obtained pure enzyme, after lyophilization, can be directly used in the preparation of blood glucose test strips or sensors without the need for additional FAD, and its specific activity can reach 592-836 U / mg solid. The scope of protection of this application covers all processes for fermenting glucose dehydrogenase using the recombinant engineered strain of this application, regardless of the specific culture conditions (e.g., temperature, pH, inducer concentration, time), whether riboflavin is added to the culture medium, or how the purification process changes. As long as the above two core steps of culture and recovery are included, and the produced enzyme has the sequence of SEQ ID NO:1 or SEQ ID NO:2, it falls within the scope of protection.

[0033] The embodiments of this application also provide Aspergillus niger strains for producing glucose dehydrogenase ( Aspergillusniger This strain was deposited on May 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 42719. This strain is a wild-type strain with endogenous glucose dehydrogenase activity, isolated and screened by the inventors from natural soil samples. Its genome naturally contains a glucose dehydrogenase gene encoding the amino acid sequence shown in SEQ ID NO: 1. As a natural producer of the wild-type glucose dehydrogenase shown in SEQ ID NO: 1, this strain, under suitable culture medium and conditions, can synthesize and secrete naturally active glucose dehydrogenase protein using its own endogenous metabolic pathway during fermentation. After collecting the fermentation broth and removing the bacterial cells, the enzyme protein can be obtained through conventional protein purification steps. Using this as a starting material, the inventors conducted in vitro directed evolution of the gene using error-prone PCR random mutagenesis technology, followed by recombinant expression and functional screening, ultimately obtaining a mutant glucose dehydrogenase with significantly enhanced thermostability and catalytic activity, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0034] In practical production, the *Aspergillus niger* strain can be inoculated into a liquid culture medium containing a carbon source (such as glucose) and a nitrogen source, and cultured under shaking or deep aeration conditions suitable for the growth of filamentous fungi. This allows the fungal cells to proliferate rapidly and secrete glucose dehydrogenase extracellularly. After fermentation, the mycelium and solid residue are removed by centrifugation or filtration, and the clarified fermentation supernatant containing the target enzyme protein is collected. This supernatant can be further purified using protein purification techniques known in the art (such as one or more combinations of salting out, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, or gel filtration chromatography) to obtain high-purity glucose dehydrogenase (SEQ ID NO: 1), and can be further mutated to obtain the high-performance mutant shown in SEQ ID NO: 2.

[0035] This application also provides the application of glucose dehydrogenase in the preparation of blood glucose test strips. The blood glucose test strip is an important component of clinical or home blood glucose meters, and its core functional layer contains components such as enzymes, electron mediators, and stabilizers. Specifically, the glucose dehydrogenase of SEQ ID NO:1 or SEQ ID NO:2 is immobilized in the reaction area of ​​the test strip. The specific method of using the test strip is as follows: The user adds a capillary blood sample (usually 2-5 μL) to the sample application area of ​​the test strip. The blood sample enters the reaction layer through capillary action; the glucose in the sample is oxidized to gluconate-δ-lactone under the catalysis of glucose dehydrogenase, while the FAD cofactor in the enzyme molecule is reduced to FADH2. The reduced enzyme transfers electrons to electron mediators (such as potassium ferricyanide, PMS, etc.) in the reaction layer. After reduction, the mediator diffuses to the electrode surface and loses electrons under a constant potential, generating a current signal. This current intensity is directly proportional to the glucose concentration in the sample, and the blood glucose concentration value is automatically calculated and displayed by an electrochemical workstation or the current-concentration standard curve built into the blood glucose meter. The entire reaction process can be completed within 30 seconds. Because the enzyme in this application uses FAD as a coenzyme and does not rely on oxygen molecules as electron acceptors, the measurement results are not affected by the dissolved oxygen concentration in the blood sample, avoiding errors caused by changes in oxygen partial pressure in traditional glucose oxidase test strips. Simultaneously, the enzyme's high specificity for glucose (cross-reactivity with xylose, maltose, etc., less than 1%) allows it to eliminate the influence of common interfering sugars in test strip applications, ensuring the accuracy of the test results. This application protects the use of this enzyme in the preparation of blood glucose test strips, including but not limited to all methods of coating the enzyme alone or in combination with other auxiliary components (such as buffers, stabilizers, surfactants) onto the test strip substrate, as long as the test strip uses the glucose dehydrogenase of this application as the core recognition element, it is covered within the scope of protection of this application.

[0036] This application also provides the application of glucose dehydrogenase in the preparation of a blood glucose sensor. The blood glucose sensor typically refers to a portable detection device based on electrochemical principles, the core of which is a biosensitive layer containing an enzyme capable of specifically recognizing glucose. In this application, the glucose dehydrogenase of SEQ ID NO: 1 or SEQ ID NO: 2 is immobilized on the surface of the working electrode using cross-linking, embedding, or covalent bonding methods, and combined with a reference electrode (such as an Ag / AgCl electrode) and a counter electrode (such as a platinum wire electrode) to construct a three-electrode enzyme electrode sensor. The specific method of using this sensor is as follows: First, a constant potential (usually +0.3V) is applied between the working electrode and the reference electrode using a potentiostat. After the background current stabilizes, the blood sample to be tested is dropped into the working area of ​​the sensor. The glucose in the sample undergoes an enzymatic reaction with the glucose dehydrogenase on the electrode surface. The electrons generated by the reaction are transferred to the working electrode via a mediator, generating an oxidation current. The steady-state current value within 10-60 seconds after the reaction starts is recorded using a chronoamperometry method. By comparing this value with a pre-established standard curve (current value - glucose concentration), the blood glucose concentration can be calculated. In continuous monitoring sensors, the sensor can come into contact with tissue fluid through a microdialysis probe or subcutaneous implantation, and record the current signal every 1-5 minutes to achieve real-time dynamic monitoring of blood glucose concentration.

[0037] The enzyme described in this application exhibits significant advantages in sensor applications: First, its high thermal stability (virtually no loss of activity after 20 minutes of treatment at 60°C) eliminates the need for stringent cold chain conditions during storage and transportation, extending shelf life. Second, the enzyme requires no external coenzyme, simplifying sensor manufacturing processes and reducing costs. Third, its low substrate cross-reactivity (relative reactivity of D-xylose is only 0.6%) significantly improves sensor selectivity, making it particularly suitable for clinical scenarios with extremely high accuracy requirements, such as intensive care, newborn screening, and post-xylose loading tests. This embodiment covers all sensor types utilizing the glucose dehydrogenase described in this application as a molecular recognition element, including disposable electrochemical test strip sensors, implantable continuous monitoring sensors, and microfluidic chip integrated sensors. As long as the enzyme described in this application is used in its preparation process, it falls within the scope of protection.

[0038] The following is a description of a specific embodiment.

[0039] Example 1: GDH-AF shake-flask fermentation and purification 1.1 Fermentation Culture Strain activation: Aspergillus flavus (ATCC 200026) strain was inoculated onto Czapek agar slant and cultured at 30 ℃ for 5-7 days. The spores were washed off with sterile physiological saline to prepare a spore suspension.

[0040] Seed culture: Inoculate the spore suspension into potato glucose liquid medium (containing 20 g / L glucose) at a 5% inoculum and culture at 30 ℃ and 180 rpm for 48 h.

[0041] Fermentation for enzyme production: Take the seed culture and transfer it to fresh fermentation medium (same as seed culture medium) at an inoculation rate of 10%, and incubate at 30 ℃ and 200 rpm for 72-96 h.

[0042] Collect the supernatant: Filter the fermentation broth with gauze and collect the supernatant.

[0043] 1.2 Purification method: Ammonium sulfate precipitation: Slowly add ammonium sulfate to the supernatant until 60% saturation, stir at 4 °C for 30 min, centrifuge at 12,000 × g for 30 min, take the precipitate and dissolve it in a small amount of 20 mM Tris-HCl (pH 7.5), dialyze against this buffer overnight.

[0044] Ion exchange chromatography: The dialysate was loaded onto a Q-Sepharose anion exchange column (equilibrated with 20 mM Tris-HCl at pH 7.5), eluted with a linear gradient of 0-0.5 M NaCl, and the GDH-AF activity peak (at approximately 0.2-0.3 M NaCl) was collected.

[0045] Gel filtration chromatography: Combine the active components, concentrate them, and load them onto a Superdex 200 column. Elute isocratically with 50 mM sodium phosphate buffer (pH 7.0), collect the GDH-AF activity peak, and obtain the purified enzyme. Freeze-dry the purified enzyme for use as a control.

[0046] Example 2: Construction of a vector for glucose dehydrogenase and shake-flask fermentation 2.1 Experimental Materials: pPIC9K vector plasmid and X-33 bacterial strain were purchased from Thermo Fisher Scientific; YNB (yeast nitrogen source) was purchased from Beijing Tiangen Biotech Co., Ltd.; peptone and yeast extract were purchased from OXOID; biotin was purchased from Sigma; Ni column and SP column were purchased from GE; Sac I enzyme and enzyme digestion buffer were purchased from NEB (Beijing) Co., Ltd. 2.2 Construction of yeast expression of glucose dehydrogenase protein: A strain of Aspergillus niger with glucose dehydrogenase activity was obtained by isolating it from soil. AspergillusnigerAfter gene sequencing and codon optimization, the strain was synthesized by Qingke Biotechnology. EcoRI and SalI restriction sites were reserved at the N-terminus and C-terminus, respectively. The target fragment and pPIC9K vector were ligated by T4 ligase after double digestion with EcoRI and SalI. The glucose dehydrogenase DNA sequence contains an 8×His tag at the C-terminus. Then, it was transformed into DH5α competent cells and plated on LLB (Low Salt LB medium) solid plates (G418 concentration 0.8 mg / ml). Colony PCR was performed on each gene to screen 12 transformants, and recombinants were identified by double digestion. The successfully constructed vector was named pPIC9K-GDH.

[0047] 2.3 Mini-prep of glucose dehydrogenase expression plasmid: A glucose dehydrogenase-positive DH5α strain was inoculated into 10 ml of LLB medium containing 50 μg / ml kanamycin. At mid-logarithmic growth (OD600 = 0.5-0.7), 0.85 ml of the bacterial culture was removed and added to sterile glycerol for preservation at -80 °C. The remaining bacteria were cultured overnight at 37 °C. Plasmids were extracted using a plasmid mini-prep kit and aliquoted for storage.

[0048] LLB (Low Salt LB) medium: Trypton 1%, Yeast Extract 0.5%, NaCl 0.5%, pH 7.5. Add 2% agar powder when preparing plates. Autoclave at 121 °C for 20 min. Can be stored at room temperature for several months. When used to culture pPIC9K prokaryotic host bacillus DH5α, allow the medium to cool to at least 55 °C, add kanamycin to a final concentration of 50 μg / ml, and it can be stored at 4 °C for 3-4 weeks.

[0049] 2.4 Linearization of glucose dehydrogenase expression plasmid: The expression plasmid pPIC9K-GDH was linearized using Sac I (209 bp) restriction enzyme. The restriction enzyme digests the 5'AOX1 site of the expression vector pPIC9K. The digestion volume was 100 μL (plasmid > 10 μg). After digestion, electrophoresis was performed to check if the plasmid was cleaved. Comparative electrophoresis was performed before and after linearization of the yeast. The cleaved bands moved slower, and the intact plasmid appeared first. If the digestion was incomplete, there would be two bands in the linearized lane. The reaction was terminated by adding EDTA after complete digestion, or by heat inactivation at 65 °C for 30 min.

[0050] 2.5 Phenol-chloroform extraction plasmids: (1) Add approximately 100 μL of enzyme digestion solution to a final volume of 400 μL; (2) Add an equal volume of phenol-chloroform (take the lower layer of phenol-chloroform), mix well, and let stand at 4 ℃ for 10 min; (3) Take the upper water sample, add 1 / 10 volume of 3 M sodium acetate and 2.5 volume of pre-cooled 100% ethanol, place at -20 ℃ for 1 h, centrifuge at 4 ℃ for 20 min, and remove the supernatant; (4) Add 250 μL of 80% ethanol to wash the DNA, centrifuge at 4 ℃ for 20 min, and discard the supernatant; (5) Dry it, add 10 μL of sterile ddH2O, and store at -20 ℃ for later use.

[0051] Preparation of competent yeast cells and electrotransfer of methanolophilic yeast: (1) After streaking the yeast, pick the clones into 5mL YPD and 50mL centrifuge tubes and incubate overnight at 30℃ and 220rpm. Take 0.25mL of seed culture and re-inoculate it into a 2L Erlenmeyer flask containing 500mL of fresh culture medium and incubate overnight.

[0052] (2) OD600=1.3-1.5, generally shake for 12h-18h, 1500 g, centrifuge at 4 ℃ for 5 min; (3) Add 500 mL of pre-cooled (0 ℃) sterile ddH2O, centrifuge at 1500 g for 5 min at 4 ℃; add 250 mL of pre-cooled (0 ℃) sterile ddH2O, centrifuge at 1500 g for 5 min at 4 ℃; (4) Resuspend the precipitate in 20 ml of ice-cold (0 ℃) 1 M sorbitol, centrifuge at 1500 g, 4 ℃ for 5 min; resuspend the precipitate in 1 ml of ice-cold (0 ℃) 1 M sorbitol, with a final volume of about 1.5 ml, and place it in an ice bath for later use. Do not preserve the cells.

[0053] (5) Take 80 μL of the above cells + linear DNA (5-10 μg), mix well and transfer into a 0.2 cm electroporation cuvette. Place on ice for 5 min, wipe the metal plate of the electroporation cuvette dry, voltage: 1500V, resistance: 400 Ω, capacitance: 25 μF, pulse time: 10mS, and start electroporation. (6) Immediately add 1 mL of ice-cold (0 °C) 1M sorbitol and transfer it to a 15 mL sterile centrifuge tube; (7) Incubate at 30℃ for 1-2 hours; (8) Take 50-200 μl and spread it on a YPDS plate, containing G418 0.8 mg / ml; (9) Transformants will grow on the plate after being placed in a 30 ℃ incubator for 2-3 days; YPD or YPDS medium preparation (Yeast Extract Peptone Dextrose Medium): yeast extract 1%, peptone 2%, dextrose 2%, ±1 M sorbitol, ±2% agar, G418 0.8 mg / ml Liquid YPD medium can be stored at room temperature and is the most basic culture medium for Pichia pastoris. Agar YPDS plates can be stored at 4°C for several months. Adding G418 0.8 mg / ml creates YPDG medium, which can be stored at 4°C for 1-2 weeks.

[0054] 2.6 Identification and trial expression of transformants: (1) Once a single colony grows on the YPDS plate, pick 10-20 single colonies and mark them on the back of the plate. Inoculate them into fresh YPD medium and culture. Take the bacteria, add glycerol and store at -80 ℃. Transfer them to a small Erlenmeyer flask containing 50 ml of BMGY and culture at 30 ℃ and 200 rpm until OD600 = 1-1.5. (2) Transfer the culture system in the small triangular flask into a 50 ml centrifuge tube, and collect the bacteria at 3000 g for 5 min; (3) Resuspend the precipitate in BMMY until OD600=0.3 (approximately 100-200 ml); (4) Transfer to a 500 ml Erlenmeyer flask, start induction culture at 30 ℃ and 200 rpm, take out 1 ml at intervals, freeze and store at -80 ℃, and add methanol to a final concentration of 0.5%. Sampling time points: 0 h, 24 h, 48 h, 72 h, 96 h; (5) Centrifuge the samples taken at each time point for 1 min and take the supernatant. Take 30 μl of the supernatant, add 10 μl of sample loading buffer, boil for 10 min, centrifuge (13000 rpm, 5 min), and take 15 μl to run SDS-PAGE. The BMGY medium formulation is as follows: yeast extract 1% (w / v), peptone 2% (w / v), potassium phosphate buffer (pH 6.0) 100 mmol / L, YNB (yeast nitrogen basal) 1.34% (w / v), biotin 4×10⁻⁶ -5 % (w / v), glycerol 1% (w / v).

[0055] Before inducing expression of Pichia pastoris, the culture medium is sterilized by filtration of YNB and Biotin. After culturing for 24 hours, it is generally left to stand overnight to allow the yeast to settle. Then, the BMGY medium is discarded and replaced with BMMY medium to begin the induction expression stage.

[0056] BMMY medium: Yeast extract 1%, Peptone 2%, Potassium phosphate buffer (pH 6.0) 100 mmol / L, YNB 1.34%, Biotin (4 × 10⁻⁶) -5 )%, methanol 3% Pichia pastoris expression induction medium was sterilized by YNB and Biotion filtration. During shake-flask culture, 3% methanol was added every 24 hours for induction, typically for 72 hours.

[0057] 2.7 Shake-flask fermentation of pPIC9K-GDH strain: The constructed yeast strain expressing glucose dehydrogenase protein was inoculated into 10 ml of YPD medium and cultured with shaking at 150 rpm and 29 ℃ for 24 h to reach the logarithmic growth phase, serving as the primary seed. The primary seed was then inoculated into 100 ml of BMGY medium at a ratio of 1:100 and cultured with shaking at 150 rpm and 29 ℃ for 4 days. 1.8% (v / v) methanol was added daily to induce expression, and fermentation was stopped on the 7th day. The fermentation broth was transferred to a centrifuge and centrifuged at 12000 g for 15 min. The supernatant was collected to obtain the crude enzyme solution.

[0058] 2.8 Purification: A small sample of the collected supernatant was used for fermentation broth activity testing. The remainder was purified, with glucose dehydrogenase extracted by nickel column affinity chromatography.

[0059] Example 3: Determination of glucose dehydrogenase activity GDH enzyme activity: One unit of enzyme activity is defined as the amount of enzyme required to convert 1 μmol of D-glucose into D-glucone at 37 °C per minute under the conditions specified by the assay method.

[0060] Reaction formula: PMS stands for Phenazine methosulfate. DCIP stands for 2,6-Dichloroindophenol. This assay is based on the decrease in absorbance at 600 nm during DCIP reduction in the following reaction.

[0061] Reagent preparation: GDH enzyme activity assay steps: Please accurately measure 1.8 ml of the reaction mixture and 20 μl of the enzyme solution into a small test tube. Add 100 μl of 20 mM PMS solution, mix well, and pre-incubate at 37 ℃.

[0062] For the blank test, add 20 μl of enzyme dilution buffer at this time.

[0063] After 3 minutes, add 100 μl of 2.0 mM DCIP solution, mix well, and start the reaction at 37 °C.

[0064] After the reaction is initiated, the rate of increase in absorbance at 600 nm per minute is measured. This rate must be measured within the linear range of the absorbance curve.

[0065] △A / min=(As / min−Ab / min)≤0.1Abs / min, where As / min is the rate of change of sample absorbance and Ab / min is the rate of change of blank absorbance.

[0066] Enzyme solution: Accurately weigh approximately 10 mg of sample and add enzyme dilution buffer to a total volume of 20 ml. Then dilute with enzyme dilution buffer as needed to adjust to the required concentration.

[0067] GDH enzyme activity calculation: Method for calculating GDH enzyme activity units: The meanings of each symbol or number are as follows: 16.3: Millimolecular extinction coefficient (cm) of DCIP at 600 nm 2 / μmol) 2.02: Final volume (ml) 0.02: Enzyme solution volume (ml) X: Concentration of the sample in the enzyme solution (mg / ml) The crude enzyme solution from the cells was used to determine the enzyme activity. High-yielding transformants were screened. After fermentation, GDH was purified and freeze-dried, and the activity was measured again. The activity of GDH was 55% higher than that of the control enzyme GDH-AF, and the activity of the mutant GDH-mut was 41% higher than that of GDH, as shown in Table 1.

[0068] Table 1 Enzyme Activity Statistics Example 4: Effects of pH and temperature on glucose dehydrogenase activity and stability 4.1 Effect of pH on enzyme activity and stability: Enzyme detection solutions were prepared using buffers with different pH values ​​(5-9) (pH 3.0-6.0, acetate buffer, pH 5.0-9.0, phosphate buffer). Enzyme activity was measured according to the method described above to determine the optimal pH for GDH reaction. GDH activity was measured at 37 °C in buffers with pH values ​​of 5-9, and the results are as follows. Figure 1 As shown, the enzyme activity is 22% at pH 5.0. With increasing pH, the glucose dehydrogenase activity gradually increases, reaching a maximum at pH 7, and then begins to decrease. Therefore, the enzyme is active between pH 6 and 8, and the optimal pH for the enzyme's reaction is 6.5–7.5.

[0069] The enzyme solution was diluted with Britton-Robinson buffer at different pH values ​​(3-10), incubated at 25 °C for 17 h, and the residual enzyme activity was measured. A pH vs. enzyme activity curve was constructed to assess the pH stability of GDH, using the enzyme activity of GDH at the optimal pH. The results are as follows: Figure 2 As shown, glucose dehydrogenase activity gradually increased when treated at pH 3-4, had no effect on glucose dehydrogenase activity when treated at pH 4-9, and then decreased with increasing pH.

[0070] 4.2 Effect of temperature on enzyme activity and stability: Enzyme activity was measured at different temperatures in 50 mM phosphate buffer (pH 6.5) according to the method described above to determine the optimal reaction temperature for GDH. GDH activity data were obtained at 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, and 65 °C. Unlike the control enzyme, the highest activity temperature for GDH was 45 °C. Temperature-GDH activity curves were constructed, and the results are shown below. Figure 3 As shown, GDH activity increases at 25-45 ℃ and gradually decreases at 45-65 ℃.

[0071] To investigate the thermostability of the enzyme, an enzyme solution with a GDH activity of 100 U / ml was incubated in 50 mM phosphate buffer at pH 6.5 at 4 ℃, 20 ℃, 40 ℃, 50 ℃, 60 ℃, and 70 ℃ for 20 min at each temperature. Residual enzyme activity was measured, and enzyme activity curves of GDH after incubation at different temperatures for 20 min were constructed. The results are shown below. Figure 4As shown, after treatment at 4-50 ℃ for 20 min, the enzyme activity was basically not lost; after treatment at 70 ℃ for 20 min, the enzyme activity was basically lost; after treatment at 60 ℃ for 20 min, the activity of the mutant GDH-mut was basically not lost, while the activity of GDH-AF and GDH was only about 40%.

[0072] Example 5: Substrate Specificity The specificity of the three enzymes GDH-AF, GDH, and GDH-mut to different substrates was tested using the method described above. The relative activity of GDH and GDH-mut to D-Xylose was only 0.6%, which was significantly lower than that of the control enzyme GDH-AF. This helps to greatly reduce the impact of D-Xylose on false positives in blood glucose testing and improve the accuracy of clinical testing.

[0073] Table 2: Substrate Specificity Example 6: GDH Yeast Fermentation Tank Process 6.1. Shake-flask culture of seed culture Single colonies of recombinant engineered strains were picked from glycerol tubes or plates and inoculated into BMGY medium. The culture was then incubated at 30°C and 250 rpm on a shaker for 16 to 24 hours until the OD600 value of the culture reached 2 to 6, thus obtaining the seed culture.

[0074] 6.2. Preparation of Fermentation Tank The fermenter and the glycerol-containing basal salt medium were sterilized. When the medium temperature dropped to 30°C, the pH was adjusted to 5.0 using 28% ammonia. 4.35 ml of sterile filtered PTM1 salt solution was added to each liter of medium. The PTM1 salt solution was formulated as follows: CuSO4·5H2O 5 g, NaI 0.08 g, MnSO4·H2O 3 g, Na2MoO4·2H2O 0.2 g, H3BO3 0.02 g, CoCl2 0.5 g, ZnCl2 20 g, FeSO4 65 g, biotin 0.2 g, concentrated H2SO4 5 ml, and diluted to 1 L with distilled water. The solution was then filtered and sterilized before use.

[0075] 6.3. Batch fermentation of glycerol The seed culture was transferred to the fermenter at an inoculum volume of 5% to 10% of the initial fermentation volume to begin batch fermentation. During the culture, the dissolved oxygen level was maintained above 20% by adjusting the stirring speed and aeration rate. Batch fermentation lasted for 18 to 24 hours, and the dissolved oxygen level rose to nearly 100% when the glycerol was completely consumed, indicating the end of the batch fermentation phase.

[0076] 6.4. Glycerol-fed culture After the batch fermentation of glycerol was completed, fed-batch glycerol culture was initiated. A 50% (w / v) glycerol feed solution containing PTM1 salt was added to the fermenter at a rate of 12 ml of PTM1 per liter of feed solution. The flow rate was controlled at 18.15 ml / h per liter of initial fermentation broth. The fed-batch glycerol culture continued for 4 h, during which cell biomass further increased.

[0077] 6.5. Methanol-fed induction culture After the glycerol fed-batch culture was completed, the glycerol feeding was stopped, and the methanol fed-batch culture was started. A methanol feed solution containing PTM1 salt was added to the fermenter at a rate of 12 ml / L methanol. The initial feed rate was controlled at 3.5 ml / h / L of initial fermentation broth volume and maintained for 2-3 hours to allow the culture to adapt to the methanol-based growth environment. Once the culture had adapted to methanol, the feed rate was gradually increased to 7 ml / h / L of initial fermentation broth volume, maintained for 2 hours, and then increased to 10.5 ml / h, maintaining this rate until fermentation ended. Throughout the methanol fed-batch culture, dissolved oxygen was maintained above 20% by adjusting the stirring speed, aeration rate, or supplementing with pure oxygen, and the fermentation temperature was controlled at 30 °C. The methanol fed-batch culture lasted for 72 hours, with a total methanol feed volume of approximately 700 ml / L of initial fermentation broth volume.

[0078] 6.6. Harvesting of Fermentation Supernatant After fermentation, the culture medium was transferred to a centrifuge container, and the bacterial cells and supernatant were separated by centrifugation. The supernatant containing glucose dehydrogenase was collected for subsequent purification.

[0079] In summary, this application provides glucose dehydrogenases with amino acid sequences as shown in SEQ ID NO: 1 (GDH) or SEQ ID NO: 2 (GDH-mut).

[0080] The enzyme activity assay results showed that the specific activity of the control enzyme GDH-AF derived from Aspergillus flavus was 380 U / mg, while the specific activity of the proposed GDH reached 592 U / mg and the specific activity of GDH-mut reached 836 U / mg. The specific activities of the latter two were increased by 55% and 120% respectively compared with the control enzyme.

[0081] pH activity curves show that the GDH and GDH-mut of this application exhibit high activity between pH 6 and 8, with the optimal reaction pH being 6.5 to 7.5. pH stability experiments show that the enzyme activity of the GDH and GDH-mut of this application is essentially unaffected after treatment within the pH range of 4 to 9 for 17 h. Temperature activity curves show that the GDH and GDH-mut of this application possess catalytic activity within the temperature range of 25 °C to 65 °C, with the optimal reaction temperature being 45 °C.

[0082] Thermostability experiments showed that GDH enzyme activity was not significantly lost after treatment at 4 ℃ to 50 ℃ for 20 min, and some residual enzyme activity was still retained after treatment at 60 ℃ for 20 min. GDH-mut enzyme activity was not significantly lost after treatment at 60 ℃ for 20 min, while the residual enzyme activity of the control enzyme GDH-AF under the same heat treatment conditions was significantly lower than that of GDH-mut, indicating that the enzyme of this application is significantly superior to the existing FAD-GDH in terms of thermostability.

[0083] Substrate specificity assays showed that the GDH and GDH-mut of this application are highly specific for D-glucose, have no cross-reactivity with interfering sugars such as maltose, D-galactose, and D-fructose, and exhibit extremely low relative reactivity with D-xylose. In contrast, the control enzyme GDH-AF showed significant cross-reactivity with D-xylose. This indicates that the GDH and GDH-mut of this application are significantly superior to the existing FAD-GDH in terms of substrate specificity.

[0084] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. Glucose dehydrogenase, characterized in that, Glucose dehydrogenase has the amino acid sequence of SEQ ID NO:

1.

2. Glucose dehydrogenase, characterized in that, Glucose dehydrogenase has the amino acid sequence of SEQ ID NO:

2.

3. The gene encoding the glucose dehydrogenase of claim 1.

4. A recombinant vector comprising the gene of claim 3.

5. A recombinant engineered strain containing the recombinant vector of claim 4.

6. Methods for preparing glucose dehydrogenase, including: S1: Cultivate the recombinant engineered strain according to claim 5; S2: Recover the glucose dehydrogenase from the recombinant engineered strain or a culture of the recombinant engineered strain.

7. A *Aspergillus niger* strain that produces the glucose dehydrogenase of claim 1, characterized in that, The Aspergillus strain is classified as Aspergillus niger (Aspergillus niger). Aspergillus niger It was deposited on May 29, 2026 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 42719.

8. The application of the glucose dehydrogenase according to claim 1 in the preparation of blood glucose test strips.

9. The application of the glucose dehydrogenase according to claim 1 in the preparation of a blood glucose sensor.

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