WO2022151746A1 - 一种融合酶及所述融合酶在纸基生物传感器中的应用 - Google Patents

一种融合酶及所述融合酶在纸基生物传感器中的应用 Download PDF

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WO2022151746A1
WO2022151746A1 PCT/CN2021/116537 CN2021116537W WO2022151746A1 WO 2022151746 A1 WO2022151746 A1 WO 2022151746A1 CN 2021116537 W CN2021116537 W CN 2021116537W WO 2022151746 A1 WO2022151746 A1 WO 2022151746A1
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fusion enzyme
amino acid
acid sequence
paper
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公维丽
马耀宏
王丙莲
刘庆艾
郑岚
李秋顺
韩庆晔
蔡雷
孟庆军
杨艳
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山东省科学院生物研究所
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01047—Glucose 1-dehydrogenase (1.1.1.47)
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    • C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
    • C12Y101/9901—Glucose dehydrogenase (acceptor) (1.1.99.10)
    • G—PHYSICS
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/685—Aspergillus niger

Definitions

  • the invention belongs to the technical field of biological enzyme genetic engineering and biological sensing, and in particular relates to a flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) gene and a carbohydrate binding module (CBM) gene constructed by GDH-linker-CBM fusion enzyme and application of the fusion enzyme in paper-based biosensors.
  • FAD flavin adenine dinucleotide
  • GDH flavin adenine dinucleotide
  • CBM carbohydrate binding module
  • oxidoreductases can be directly immobilized into paper substrates by physical adsorption; (2) many microfluidic components can be integrated on paper with complex 3D microfluidic channels; (3) diverse electrodes Materials can be easily screen printed onto paper and do not require expensive cleanroom facilities.
  • Glucose sensors used for blood glucose monitoring usually use glucose oxidase as a biological identification element, and glucose oxidase is easily affected by the partial pressure of oxygen, so it is more limited in the detection of samples with obvious differences in oxygen content such as venous blood, arterial blood, and high altitude.
  • Glucose dehydrogenase (GDH) with flavin adenine dinucleotide (FAD) as coenzyme does not use oxygen as electron acceptor, and has high enzymatic activity, strong substrate specificity and good stability.
  • the research hotspot enzyme molecule of portable glucose sensor is usually randomly fixed on the electrode surface by physical adsorption or covalent binding. The active center of the enzyme molecule is often hidden or cannot be correctly oriented, resulting in a large loss of enzyme activity, and the detection sensitivity of the portable glucose sensor is poor.
  • Carbohydrate-Binding Modules are natural carbohydrate-active enzyme molecules that have no catalytic activity themselves, but can bind specific domains to polysaccharides such as cellulose.
  • CBM Carbohydrate-Binding Modules
  • the inventor believes that providing a paper-based sensor based on glucose dehydrogenase is helpful to obtain a blood glucose monitoring device with excellent performance, and is a promising transformation direction.
  • the present invention utilizes the specific affinity and adsorption properties of CBM to cellulose to construct a fusion enzyme, and uses a paper base as an immobilization carrier for the fusion enzyme molecule. It was determined that the optimized fusion enzyme could not only show good affinity with the paper-based carrier, but also improve the detection sensitivity of the enzyme electrode sensor from various aspects.
  • a first aspect of the present invention provides a fusion enzyme comprising a glucose dehydrogenase (GDH) peptide segment and a carbohydrate binding module (CBM) peptide segment, and the two parts are connected by a flexible peptide chain.
  • GDH glucose dehydrogenase
  • CBM carbohydrate binding module
  • the present invention obtains a fusion enzyme, which can effectively increase the binding force of glucose dehydrogenase and cellulose paper base through the connection of carbohydrate binding modules (CBM), and is expected to obtain a detection sensitivity Higher paper-based sensors.
  • CBM carbohydrate binding modules
  • the fusion enzyme obtained by the above design idea can not only obtain good stability of enzyme activity, but also the detection sensitivity of the prepared paper-based biosensor has also been significantly improved, and the biological samples include Common interfering substances including ascorbic acid, uric acid and urea will not affect the detection effect of the above sensors.
  • the application of the above fusion enzymes in the development of paper-based production sensors is expected to obtain blood glucose, urine sugar and other detection products with better detection performance.
  • the second aspect of the present invention provides the application of the fusion enzyme of the first aspect in preparing a glucose detection element.
  • the glucose detection element is further a paper-based biosensing element.
  • the paper-based glucose detection device has the advantages of simple assembly and portable use.
  • the traditional glucose oxidase detection element is affected by the partial pressure of oxygen, which has great limitations in the detection of samples with obvious differences in oxygen content, and the glucose oxidase is usually immobilized by physical adsorption or covalent binding. Instability, the active center of the enzyme molecule will be affected, and the enzyme activity will also be reduced.
  • the above fusion enzyme provided by the present invention adopts glucose dehydrogenase (GDH) with flavin adenine dinucleotide (FAD) as coenzyme as the enzyme element, which can eliminate the influence of oxygen partial pressure on the measurement result.
  • GDH glucose dehydrogenase
  • FAD flavin adenine dinucleotide
  • glucose dehydrogenase (GDH) is combined with the carbohydrate binding module, a better binding ability can be obtained with the paper base, which effectively reduces the probability that the traditional glucose oxidase is detached from the carrier and the enzyme activity is reduced.
  • a third aspect of the present invention provides a paper-based biosensing element, wherein the sensing element is cellulose paper immobilized with the fusion enzyme of the first aspect.
  • a fourth aspect of the present invention provides a blood glucose detection kit, which includes the immobilized carrier of the fusion enzyme of the first aspect and/or the paper-based biosensor element of the third aspect.
  • fusion enzymes are constructed by using the specific affinity and adsorption properties of CBM to cellulose, and paper bases are used as immobilization carriers of fusion enzyme molecules to improve the detection sensitivity of enzyme electrode sensors.
  • the present invention provides the excellent performance of the fusion enzyme for the first time. Based on the discovery of the cellulose binding specificity of the fusion enzyme, those skilled in the art can easily obtain a variety of glucose level monitoring products with better performance. Monitoring and prevention and treatment of diabetes are of great significance.
  • FIG 1 shows the detection of heterologously expressed glucose dehydrogenase by SDS-PAGE
  • (a) is the predicted wild-type glucose dehydrogenase (GDH) structure
  • (b), (c), (d) are the predicted fusion glucose dehydrogenase (GDH-NL-CBM2, GDH-(GGGGS)2-CBM2, GDH-(EAAAK)3-CBM2) structures;
  • Fig. 2 is the optimum temperature and optimum pH determination of GDH and GDH-NL-CBM2;
  • Figure 3 is a schematic diagram of a cellulose paper-based biosensor immobilized with GDH-NL-CBM2.
  • Figure 4 shows the electrochemical performance detection results of GDH and GDH-NL-CBM2/cellulose paper-based biosensors in PBS and glucose solutions with different concentrations
  • (a), (b) are the cyclic voltammetry detection curves of GDH and GDH-NL-CBM2, respectively; (c), (d) are the chronoamperometry detection curves of GDH and GDH-NL-CBM2, respectively; (e) are GDH and GDH - Linear response analysis of NL-CBM2 to glucose.
  • the paper-based glucose sensor has the advantages of portability and easy development.
  • a fusion enzyme of flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) and carbohydrate binding module (CBM) is proposed.
  • a first aspect of the present invention provides a fusion enzyme comprising a glucose dehydrogenase (GDH) peptide segment and a carbohydrate binding module (CBM) peptide segment, and the two parts are connected by a flexible peptide chain.
  • GDH glucose dehydrogenase
  • CBM carbohydrate binding module
  • the glucose dehydrogenase peptide segment is derived from Aspergillus niger An76.
  • amino acid sequence of the GDH is shown in SEQ ID NO: 1, or the amino acid sequence with 80% and above similarity with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence with the same function is also claimed in the present invention. within the range.
  • the present invention also provides the encoding gene of the amino acid sequence shown in SEQ ID NO: 1, and its sequence is shown in SEQ ID NO: 2; in addition to the gene sequence, due to the degeneracy of codons, it can also be translated to obtain SEQ ID NO: 1.
  • the gene sequence of the amino acid sequence shown in ID NO: 1, because it can achieve the same technical effect, is also within the scope of protection of the present invention.
  • the carbohydrate binding module (CBM) peptide is selected from one of CBM1, CBM2, CBM3, CBM5, and CBM10.
  • carbohydrate binding module is the second family CBM (CBM2), and the sequence of the CBM2 is as shown in SEQ ID NO:3, or has 80% or more similarity with the amino acid sequence shown in SEQ ID NO:3 , amino acid sequences with the same function are also within the scope of the claimed protection of the present invention.
  • the present invention also provides the encoding gene of the amino acid sequence shown in SEQ ID NO: 3, and its sequence is shown in SEQ ID NO: 4; in addition to the gene sequence, due to the degeneracy of codons, it can also be translated to obtain SEQ ID NO: 3.
  • the gene sequence of the amino acid sequence shown in ID NO: 3, because it can achieve the same technical effect, is also within the scope of protection of the present invention.
  • the flexible peptide chain is a connecting peptide
  • the connecting peptide is selected from endo- ⁇ -xylanase (EM_PRO: Z81013.1) in the genome of (EAAAK)3, (GGGGS)2 Thermobifida fusca One or any of the natural linker sequences (NL) connecting the catalytic domain and the CBM2 domain.
  • the connecting peptide is selected from the natural linker connecting the catalytic domain and the CBM2 domain in the endo- ⁇ -xylanase (EM_PRO: Z81013.1) in the Thermobifida fusca genome. sequence (NL). It has been verified that the fusion expression of GDH and Carbohydrate-Binding Modules (CBM) by adding connecting peptides can directly bind GDH to cellulose paper materials, and can make the GDH active center not hidden and maintain high activity. , thereby ensuring the detection sensitivity of the glucose sensor.
  • CBM Carbohydrate-Binding Modules
  • the present invention also provides the connecting peptide (EAAAK) 3 shown in SEQ ID NO:7 and the corresponding encoding gene (SEQ ID NO:8), the connecting peptide (GGGGS) shown in SEQ ID NO:9 2 and its corresponding coding gene (SEQ ID NO: 10).
  • the natural connecting peptide sequence of the CBM2 structural domain is shown in SEQ ID NO:5, or the amino acid sequence with 80% or more similarity with the amino acid sequence shown in SEQ ID NO:5, and the same function is also in this within the scope of the claimed invention.
  • the present invention also provides the coding gene of the amino acid sequence shown in SEQ ID NO: 5, and its sequence is shown in SEQ ID NO: 6; in addition to the gene sequence, due to the degeneracy of codons, it can also be translated to obtain SEQ ID NO: 6.
  • the gene sequence of the amino acid sequence shown in ID NO: 5, because it can achieve the same technical effect, is also within the scope of protection of the present invention.
  • the fusion enzyme is from the N-terminus to the C-terminus, and its amino acid sequence is shown in SEQ ID NO: 11.
  • the present invention also provides the encoding gene of the above-mentioned fusion protein, and the encoding gene sequence of the fusion protein is as shown in SEQ ID NO:12.
  • fusion enzyme GDH-(EAAAK)3-CBM2 amino acid sequence such as SEQ ID NO: 13
  • fusion enzyme GDH-(GGGGS)2- CBM2 amino acid sequence such as SEQ ID NO: 15
  • SEQ ID NO: 16 amino acid sequence such as SEQ ID NO: 16
  • the expression method of the above fusion enzyme can be achieved by linking the polynucleotide to a vector or by integrating it into the host cell genome.
  • the term "vector” refers to a DNA construct comprising a nucleotide sequence encoding a desired protein operably linked to appropriate expression control sequences for expression of the desired protein in a suitable host cell.
  • the regulatory sequences include a promoter that can initiate transcription, an optional operator sequence that regulates transcription, a sequence encoding an appropriate mRNA ribosomal binding site, and a sequence that regulates transcriptional and translational termination.
  • the present invention provides a specific preparation method, namely transforming the encoded gene into Pichia pastoris, and then performing the steps of expression and purification.
  • the vector used in the present invention is not particularly limited as long as it can replicate in a host cell, and any known vector in the art can be used.
  • conventional vectors may include native or recombinant plasmids, cosmids, viruses and phages.
  • pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Karon 4A and Karon 21A can be used as phage vectors or cosmid vectors.
  • pBR type, pUC type, pBluescriptII type, pGEM type, pTZ type, pCL type and pET type can be used as the plasmid vector.
  • the vector usable in the present invention is not particularly limited, and any known expression vector can be used.
  • pDZ, pPIC9k, pACYC184, pCL, pECCG117, pUC57, pBR322, pMW118 or pCC1BAC vectors can be used.
  • the second aspect of the present invention provides the application of the fusion enzyme of the first aspect in preparing a glucose detection element.
  • the glucose detection element is a paper-based biosensing element.
  • a third aspect of the present invention provides a paper-based biosensing element, wherein the sensing element is cellulose paper immobilized with the fusion enzyme of the first aspect.
  • the preparation method of the paper-based biosensing element is as follows: the fusion enzyme is immobilized on a cellulose paper base, and then electrodes are printed on the paper base with the fusion enzyme immobilized.
  • the preparation method is as follows: the polydimethylsiloxane and lanolin are mixed by ultrasonic and then printed on the nanocellulose paper to form a hydrophobic area, and a hydrophobic area is formed in the hydrophilic area of the nanofiber paper.
  • the fusion enzyme described in the first aspect is dropped in the printing area of the working electrode, dried at room temperature, and then the working electrode, the counter electrode and the reference electrode are respectively printed in the hydrophilic area.
  • a fourth aspect of the present invention provides a blood glucose detection kit, which includes the immobilized carrier of the fusion enzyme of the first aspect and/or the paper-based biosensor element of the third aspect.
  • the blood glucose detection kit further includes a blood sample collection device and the like.
  • Embodiment 1 the fusion expression of CBM and GDH
  • CBM1, CBM2, CBM3, CBM5, and CBM10 families with binding specificity to cellulose were selected, and the amino acid frequencies and functions of different CBMs were analyzed using structural bioinformatics analysis tools (SWISS-MODEL, ClustalX, VMD and PyMO1 software). Statistical analysis was performed on information such as the architecture sequence spectrum, and CBM2 in Thermobifida fusca was screened for fusion enzyme construction.
  • the fusion enzyme molecule linker sequences (GGGGS)2, (EAAAK)3) were selected from the LinkerDB database, and the endo- ⁇ -xylanase (EM_PRO:Z81013.
  • the natural linker sequence of the catalytic domain and the CBM2 structural domain is connected in the middle; as a connecting peptide for the construction of the GDH fusion enzyme, the natural linker sequence is "LGGDSSGGGPGEPGGPGGPGEPGGPGGPGEPGGPGDGT", and the predicted GDH and GDH fusion enzyme structures are shown in Figure 1a- 1d shown.
  • the pPIC9k-GDH plasmid obtained in the previous study linked with the codon-optimized Aspergillus niger An76 GDH encoding gene was used as a template to design primers to obtain the gene encoding GDH
  • the plasmid pUC57-linker-CBM2 DNA was used as a template to design primers to obtain the encoding linker and CBM2 gene.
  • Three kinds of GDH fusion enzyme genes with different linking peptides were obtained according to the method of Nanjing Nuowei gene recombination kit.
  • the three kinds of GDH fusion enzyme genes were expressed by using pPIC9K plasmid as expression vector and Pichia pastoris GS115 as expression host to realize GDH fusion enzyme. Express.
  • PCR amplification was performed to obtain different homologous genes.
  • the GDH gene of the recombination arm was amplified by PCR using the pUC57-linker-CBM2 gene as a template and P3, P4, P3', P4', P3", and P4" as primers to obtain the coding genes of different linker peptides and CBM2.
  • PCR reactions were performed in a 50 ⁇ L system (refer to Max Super-Fidelity DNA Polymerase), the reaction conditions were pre-denaturation at 95°C for 3 min to start the cycle, denaturation at 95°C for 15s, annealing at 60°C for 15s, extension at 72°C for 1min, a total of 30 cycles, and then extension at 72°C for 5min.
  • the PCR fragments of GDH gene and linker-CBM2 gene were obtained by amplification respectively, and recovered by gel tapping (purified by product purification kit).
  • Nanjing Novizan The One Step Cloning Kit product connects the GDH gene and linker-CBM2 gene PCR fragments recovered to the pPIC9k plasmid vector, and the reaction system is 10 ⁇ L (1 ⁇ L linearized vector, 2 ⁇ L GDH gene PCR fragment, 1.5 ⁇ L linker-CBM1 gene PCR fragment, 5 ⁇ L 2 ⁇ ClonExpress Mix, 0.5 ⁇ L ddH 2 O), after pipetting and mixing, react at 50°C for 15 min, and immediately place on ice to cool to obtain the recombinant product of pPIC9k-GDH-linker-CBM2 fusion enzyme gene.
  • the recombinant product of the pPIC9k-GDH-linker-CBM2 fusion enzyme gene was mixed with Escherichia coli DH5 ⁇ , spread on 100ug/mL ampicillin-resistant LB agar plates after heat shock for 90s, and cultured at 37°C overnight. Pick a single colony, then extract the plasmid for electrophoresis, and store the plasmid at -20°C. Then use EcoRI and NotI to digest the target fragment, then send the bacterial suspension to the company for sequencing, and transform the correctly sequenced plasmid into E. coli by the same method to achieve plasmid enrichment.
  • the crude enzyme solutions of GDH fusion enzymes linked by three linking peptides were mixed with fillers containing nickel, and the mixture was rotated and combined in a refrigerator at 4°C for 6 h. Then, the target protein was eluted with a gradient concentration of imidazole solution, and the protein solution eluted with 20mM imidazole was ultrafiltered with a 3K ultrafiltration tube and a pH 5.0 disodium hydrogen phosphate-citrate buffer, at 4900rpm, 4°C, until the flow down The pH of the buffer was 5.0, the ultrafiltration was stopped, the GDH fusion enzyme solution obtained by ultrafiltration was collected, and the enzyme activity assay and SDS-PAGE were performed to detect the target protein.
  • the polydimethylsiloxane and lanolin were mixed in a ratio of 10:1, 60 Hz ultrasonic mixing for 30 min, and printed on the prepared nanocellulose paper with a pore size of 0.1 ⁇ m to form a hydrophobic area.
  • the GDH wild-type enzyme molecule (10U, 30 ⁇ L), 25% glutaraldehyde (2 ⁇ L), 0.5% chitosan (8 ⁇ L) isolated and purified in the previous research in the laboratory was dropped on the working electrode printing area of the hydrophilic area of the nanofiber paper.
  • the mixed solution and 40 ⁇ L of GDH-NL-CBM2 fusion enzyme (10 U) were dried at room temperature for 4 h.
  • the paper-based enzyme sensing element was sequentially subjected to pH 7.0 PBS buffer and different concentrations of glucose solutions (5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 50mM) for cyclic voltammetry scanning detection.
  • the results show that both paper-based enzyme sensing elements can detect a pair of obvious redox peaks of ferrocene in PBS buffer, and the current value corresponding to the oxidation peak potential (+0.26V) increases with the increase of glucose concentration. The high increases gradually, while the current value corresponding to the reduction peak potential (+0.17 V) gradually decreases (Fig.
  • the GDH-NL-CBM2 fusion enzyme paper-based sensing element Compared with the GDH sensing element, the GDH-NL-CBM2 fusion enzyme paper-based sensing element has the characteristics of simple fabrication and better detection performance. Therefore, it has a broad application prospect in the field of biosensors.

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Abstract

一种融合酶及所述融合酶在纸基生物传感器中的应用。一种黄素腺嘌呤二核苷酸(FAD)依赖型葡萄糖脱氢酶(GDH)基因与碳水化合物结合模块(CBM)基因构建的GDH-linker-CBM融合酶，通过碳水化合物结合模块及连接肽的修饰作用，将其制备成为一种纸基传感器。

Description

一种融合酶及所述融合酶在纸基生物传感器中的应用 技术领域
本发明属于生物酶基因工程和生物传感技术领域，具体涉及一种黄素腺嘌呤二核苷酸(FAD)依赖型葡萄糖脱氢酶(GDH)基因与碳水化合物结合模块(CBM)基因构建的GDH-linker-CBM融合酶以及所述融合酶在纸基生物传感器中应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
严格控制生理血糖浓度是糖尿病患者避免危及生命的并发症的唯一途径，因此血糖监测是糖尿病管理的重要组成部分。目前在医疗保健基础设施完善的发达国家，通过血糖自我监测进行血糖控制已成为对公众经济、有效的途径，但在许多资源匮乏的国家，由于缺乏实验室基础设施和训练有素的专业人员，许多致命疾病都无法应付，糖尿病护理通常得不到优先考虑。因此，开发简单、低成本、易于操作、便携式的葡萄糖传感器成为迫切需要。近年来，基于纸张的微流体体系在开发简单、低成本、便携式的葡萄糖检测设备中受到了相当多的关注。其优势包括：(1)氧化还原酶可以通过物理吸附直接固定到纸基质中；(2)许多微流体元件可以集成在具有复杂的3D微流体通道的纸上；(3)多种多样的电极材料可以很容易地通过丝网印刷到纸上，且不需要昂贵的洁净室设施。
用于血糖监测的葡萄糖传感器通常采用葡萄糖氧化酶作为生物识别元件，而葡萄糖氧化酶易受氧分压的影响，在检测静脉血、动脉血、高海拔等氧气含量差异明显的样本局限性较大。以黄素腺嘌呤二核苷酸(FAD)为辅酶的葡萄糖脱氢酶(GDH)不以氧气为电子受体，且酶活性高、底物专一性强、稳定性好，成为用于开发便携式葡萄糖传感器的研究热点酶分子。但是目前GDH通常是利用物理吸附或共价结合等方式随机固定在电极表面，酶分子活性中心常被隐藏掉或不能正确取向，导致酶活大量损失，便携式葡萄糖传感器检测灵敏度较差。
近年来，利用生物亲和吸附进行酶的固定化引起越来越多的关注，其最大优势是酶分子可与载体材料特异性结合、固定化方向可控以及酶分子构象改变最小。碳水化合物结合模块(Carbohydrate-Binding Modules，CBM)是天然碳水化合物活性酶分子，自身没有催化活性，但能对纤维素等多糖具有结合特异性的结构域。随着分子生物学、合成生物学、计算机辅助模拟等技术发展，目前研究者利用融合DNA技术试图获得多种多样CBM与酶分子融合蛋白， 以此提高酶分子与底物亲和力、酶分子稳定性或酶活。但利用CBM对纤维素的特异亲和吸附特性构建融合酶，并以纸基作为融合酶分子固定化载体，提高酶电极传感器检测灵敏性的改造策略目前国内外几乎未见报道。
发明内容
针对以上研究现状，发明人认为，提供一种基于葡萄糖脱氢酶的纸基传感器有助于获得一种性能优良的血糖监测设备，是一种前景良好的改造方向。基于该研究思路，本发明利用CBM对纤维素的特异亲和吸附特性构建融合酶，并以纸基作为融合酶分子固定化载体。经测定，该优化后的融合酶不仅能够与纸基载体表现出良好的亲和力，还能够从多个方面提高酶电极传感器的检测灵敏性。
基于上述技术效果，本发明提供以下技术方案：
本发明第一方面，提供一种融合酶，所述融合酶包括葡萄糖脱氢酶(GDH)肽段和碳水化合物结合模块(CBM)肽段，两部分通过柔性肽链连接。
基于上述设计思路，本发明得到了一种融合酶，通过碳水化合物结合模块(CBM)的连接作用，能够有效的增加葡萄糖脱氢酶与纤维素纸基的结合力，有望获得一种检测灵敏性更高的纸基传感器。进一步的，经本发明的验证，上述设计思路获得的融合酶不仅能够获得良好的酶活力稳定性，其制备得到的纸基生物传感器的检测灵敏性也得到了明显的提升，并且生物样品中包括抗坏血酸、尿酸、尿素在内的常见干扰物均不会影响上述传感器的检测效果。基于该研究结果，将上述融合酶应用于纸基生产传感器的开发，有望获得检测性能更加优良的血糖、尿糖等检测产品。
本发明第二方面，提供第一方面所述融合酶在制备葡萄糖检测元件中的应用。
所述葡萄糖检测元件进一步的为纸基生物传感元件。
纸基葡萄糖检测设备具有组装简单，使用便携的优点。传统的葡萄糖氧化酶检测元件受氧分压的影响，对氧气含量差异明显的样本检测具有很大局限性，并且葡萄糖氧化酶通常采用物理吸附或共价结合的方式进行固定，这种固定方式并不稳定，会导致酶分子活性中心受到影响，酶活也随之降低。而本发明提供的上述融合酶，采用黄素腺嘌呤二核苷酸(FAD)为辅酶的葡萄糖脱氢酶(GDH)作为酶元件，可以排除氧分压对测定结果的影响。另外，上述葡萄糖脱氢酶(GDH)与碳水化合物结合模块结合后与纸基能够获得更好的结合能力，有效的降低了传统葡萄糖氧化酶与载体脱离、酶活降低的概率。
本发明第三方面，提供一种纸基生物传感元件，所述传感元件为固定第一方面所述融合酶的纤维素纸。
本发明第四方面，提供一种血糖检测试剂盒，所述试剂盒中包括第一方面所述融合酶的固定化载体和/或第三方面所述纸基生物传感元件。
以上一个或多个技术方案的有益效果是：
目前利用CBM对纤维素的特异亲和吸附特性构建融合酶，并以纸基作为融合酶分子固定化载体，提高酶电极传感器检测灵敏性的改造策略目前国内外几乎未见报道。本发明首次提供了该融合酶的优异性能，基于该融合酶对纤维素结合特异性的发掘，本领域技术人员可以方便获得多种性能更加优良的葡萄糖水平监测产品，这对临床样品中葡萄糖水平监测及糖尿病的防治等均具有重要意义。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1为SDS-PAGE检测异源表达的葡萄糖脱氢酶；
其中(a)为预测的野生型葡萄糖脱氢酶(GDH)结构；
(b),(c),(d)为预测的融合葡萄糖脱氢酶(GDH-NL-CBM2,GDH-(GGGGS)2-CBM2,GDH-(EAAAK)3-CBM2)结构；
(c)条带M:蛋白标准物，20mM咪唑洗脱GDH的SDS-PAGE检测图谱；
(d)10mM咪唑洗脱GDH-NL-CBM2的SDS-PAGE检测图谱；
(e)超滤浓缩GDH和GDH-NL-CBM2的SDS-PAGE检测图谱。
图2为GDH和GDH-NL-CBM2最适温度、最适pH测定；
(a)为最适pH测定结果图；(b)为最适温度测定结果图。
图3为固定GDH-NL-CBM2的纤维素纸基生物传感器模式图。
图4为GDH和GDH-NL-CBM2/纤维素纸基生物传感器在PBS和不同浓度葡萄糖溶液中的电化学性能检测结果；
(a),(b)分别为GDH和GDH-NL-CBM2循环伏安检测曲线；(c),(d)分别为GDH和GDH-NL-CBM2计时电流检测曲线；(e)为GDH和GDH-NL-CBM2对葡萄糖的线性响应分析。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发 明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
正如背景技术所介绍的，纸基葡萄糖传感器具有便携、开发简便的优势，为了提供一种以黄素腺嘌呤二核苷酸(FAD)为辅酶的葡萄糖脱氢酶的纸基葡萄糖传感器，本发明提出了一种黄素腺嘌呤二核苷酸(FAD)依赖型葡萄糖脱氢酶(GDH)与碳水化合物结合模块(CBM)结合的融合酶。
本发明第一方面，提供一种融合酶，所述融合酶包括葡萄糖脱氢酶(GDH)肽段和碳水化合物结合模块(CBM)肽段，两部分通过柔性肽链连接。
优选的，所述葡萄糖脱氢酶肽段来源于Aspergillus niger An76。
进一步的，所述GDH的氨基酸序列如SEQ ID NO:1所示，或与SEQ ID NO:1所示氨基酸序列具有80％及以上相似度、具有同样功能的氨基酸序列也在本发明请求保护的范围之内。
更进一步的，本发明同时还提供SEQ ID NO:1所示氨基酸序列的编码基因，其序列如SEQ ID NO:2所示；除该基因序列外，由于密码子简并性同样能够翻译得到SEQ ID NO:1所示氨基酸序列的基因序列，由于能够实现同样的技术效果，也在本发明请求保护范围之内。
优选的，所述碳水化合物结合模块(CBM)肽段选自CBM1、CBM2、CBM3、CBM5、CBM10中的一种。
进一步的，所述碳水化合物结合模块为第2家族CBM(CBM2)，所述CBM2的序列如SEQ ID NO:3所示，或与SEQ ID NO:3所示氨基酸序列具有80％及以上相似度、具有同样功能的氨基酸序列也在本发明请求保护的范围之内。
更进一步的，本发明同时还提供SEQ ID NO:3所示氨基酸序列的编码基因，其序列如SEQ ID NO:4所示；除该基因序列外，由于密码子简并性同样能够翻译得到SEQ ID NO:3所示氨基酸序列的基因序列，由于能够实现同样的技术效果，也在本发明请求保护范围之内。
优选的，所述柔性肽链为一种连接肽，所述连接肽选自(EAAAK)3、(GGGGS)2 Thermobifida fusca基因组中内切-β-木聚糖酶(EM_PRO:Z81013.1)中连接催化结构域、CBM2结构域的天然linker序列(NL)中的一种或任意几种。
经本发明验证，效果较好的方案中，所述连接肽选自Thermobifida fusca基因组中内切-β-木聚糖酶(EM_PRO:Z81013.1)中连接催化结构域和CBM2结构域的天然linker序列(NL)。经验证，通过增加连接肽使GDH和碳水化合物结合模块(Carbohydrate-Binding Modules，CBM)融合表达可以使GDH直接结合到纤维素纸质材料上，并且可以使GDH活性 中心不被隐藏，保持高活性，从而保证了葡萄糖传感器的检测灵敏性。
另外的一些实施方式中，本发明同时还提供SEQ ID NO:7所示连接肽(EAAAK)3及对应的编码基因(SEQ ID NO:8)、SEQ ID NO:9所示连接肽(GGGGS)2及其对应的编码基因(SEQ ID NO:10)。
进一步的，所述CBM2结构域的天然连接肽序列如SEQ ID NO:5所示，或与SEQ ID NO:5所示氨基酸序列具有80％及以上相似度、具有同样功能的氨基酸序列也在本发明请求保护的范围之内。
更进一步的，本发明同时还提供SEQ ID NO:5所示氨基酸序列的编码基因，其序列如SEQ ID NO:6所示；除该基因序列外，由于密码子简并性同样能够翻译得到SEQ ID NO:5所示氨基酸序列的基因序列，由于能够实现同样的技术效果，也在本发明请求保护范围之内。
在上述优选技术方案的一种具体实施方式中，所述融合酶从N端到C端，其氨基酸序列如SEQ ID NO:11所示。同时，本发明还提供了上述融合蛋白的编码基因，所述融合蛋白的编码基因序列如SEQ ID NO:12所示。
另外的一些实施方式中，融合酶GDH-(EAAAK)3-CBM2(氨基酸序列如SEQ ID NO:13)及其对应的编码基因(SEQ ID NO:14)、融合酶GDH-(GGGGS)2-CBM2(氨基酸序列如SEQ ID NO:15)及其对应的编码基因(SEQ ID NO:16)。
另外，上述融合酶的表达方法可以通过将多核苷酸连接至载体或通过将其整合入宿主细胞基因组而实现。如本文所使用的，术语“载体”指的是包含编码期望蛋白质的核苷酸序列的DNA构建体，其可操作地连接至适当的表达调控序列以在适合的宿主细胞中表达期望的蛋白质。该调控序列包括可以引发转录的启动子、调控转录的任选的操纵基因(operator)序列、编码适当的mRNA核糖体结合位点的序列、和调控转录和翻译终止的序列。在载体被转化入适当的宿主细胞之后，其可以独立于宿主基因组复制或起作用，并且可以将载体整合入基因组本身。
进一步的，本发明提供了一种具体的制备方法，即将编码基因转化到毕赤酵母中，然后进行表达纯化的步骤。
在本发明中使用的载体不具体地限制，只要它能够在宿主细胞中复制，并且可以使用本领域中任何已知的载体。常规载体的实例可包括天然或重组质粒、黏粒、病毒和噬菌体。例如，pWE15、M13、MBL3、MBL4、IXII、ASHII、APII、t10、t11、卡隆4A和卡隆21A可以用作噬菌体载体或黏粒载体。作为质粒载体，可使用pBR型、pUC型、pBluescriptII型、pGEM型、pTZ型、pCL型和pET型。本发明中可使用的载体不具体地限制，并且可以使用任何已知的表达载体。优选地，可以使用pDZ、pPIC9k、pACYC184、pCL、pECCG117、pUC57、pBR322、pMW118 或pCC1BAC载体。
本发明第二方面，提供第一方面所述融合酶在制备葡萄糖检测元件中的应用。
优选的，所述葡萄糖检测元件为纸基生物传感元件。
本发明第三方面，提供一种纸基生物传感元件，所述传感元件为固定第一方面所述融合酶的纤维素纸。
优选的，所述纸基生物传感元件的制备方法如下：将所述融合酶固定到纤维素纸基上、然后在固定有融合酶的纸基上打印电极。
一种具体的实施方式中，所述制备方法如下：将聚二甲基硅氧烷和羊毛脂按混合超声混匀后打印在纳米纤维素纸上形成疏水区，在纳米纤维纸亲水区的工作电极打印区滴加第一方面所述融合酶，室温干燥，再在亲水区分别打印工作电极、对电极和参比电极。
本发明第四方面，提供一种血糖检测试剂盒，所述试剂盒中包括第一方面所述融合酶的固定化载体和/或第三方面所述纸基生物传感元件。
优选的，所述血糖检测试剂盒中，还包括血样采集装置等。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案，以下将结合具体的实施例详细说明本发明的技术方案。
实施例1、CBM与GDH的融合表达
选取CBM1、CBM2、CBM3、CBM5、CBM10家族里对纤维素具有结合特异性的CBMs序列，利用结构生物信息学分析工具(SWISS-MODEL、ClustalX、VMD及PyMOl软件)对不同CBMs的氨基酸频率、功能架构序列谱等信息进行统计分析，筛选出Thermobifida fusca中的CBM2用于融合酶构建。
基于CBM2和GDH结构特点分析，从LinkerDB数据库中选择融合酶分子linker序列((GGGGS)2、(EAAAK)3)，同时选择Thermobifida fusca基因组中内切-β-木聚糖酶(EM_PRO:Z81013.1)中连接催化结构域和CBM2结构域的天然linker序列；分别作为连接肽用于GDH融合酶的构建，所述天然linker序列为“LGGDSSGGGPGEPGGPGGPGEPGGPGGPGEPGGPGDGT”，预测的GDH和GDH融合酶结构如图1a-1d所示。
实施例2
以前期研究中获得的连接有密码子优化的Aspergillus niger An76 GDH编码基因的pPIC9k-GDH质粒为模板设计引物获得编码GDH的基因，以质粒pUC57-linker-CBM2 DNA为模板设计引物获得编码linker以及CBM2的基因。按照南京诺唯赞同源重组试剂盒方法获得3 种带有不同连接肽的GDH融合酶基因，3种GDH融合酶基因分别以pPIC9K质粒为表达载体，以Pichia pastoris GS115为表达宿主，实现GDH融合酶表达。
(1)GDH融合酶基因克隆：
利用同源重组的原理和Pichia pastoris GS115克隆位点附近序列，分别根据密码子优化的GDH基因序列和pUC57-linker-CBM2基因序列设计六对引物：
Figure PCTCN2021116537-appb-000001
以连接有密码子优化的Aspergillus niger An76 GDH编码基因的pPIC9K-GDH质粒为模板，分别以P1、P2，P1’、P2’，P1”、P2”为引物，PCR扩增获得带有不同同源重组臂的GDH基因，以pUC57-linker-CBM2基因为模板，以P3、P4，P3’、P4’，P3”、P4”为引物，PCR扩增获得不同连接肽和CBM2的编码基因。PCR反应在50μL体系(参照
Figure PCTCN2021116537-appb-000002
Max Super-Fidelity DNA Polymerase)中进行，反应条件为在95℃预变性3min开始循环，95℃变性15s，60℃退火15s，72℃延伸1min，共30个循环后，再于72℃延伸5min。分别扩增得到GDH基因和linker-CBM2基因PCR片段，割胶回收(产物纯化试剂盒纯化)。然后利用南京诺唯赞
Figure PCTCN2021116537-appb-000003
One Step Cloning Kit产品将GDH基因和linker-CBM2基因PCR割胶回收片段连接到pPIC9k质粒载体上，反应体系为10μL(1μL线性化载体，2μL  GDH基因PCR片段，1.5μL linker-CBM1基因PCR片段，5μL 2×ClonExpress Mix，0.5μL ddH 2O)，吸打混匀后，50℃反应15min，立即置于冰上冷却，即得pPIC9k-GDH-linker-CBM2融合酶基因重组产物。
(3)pPIC9k-GDH-linker-CBM2融合酶基因转化大肠杆菌富集质粒
将pPIC9k-GDH-linker-CBM2融合酶基因重组产物与大肠杆菌DH5α混合，热激90s后涂布于100ug/mL氨苄抗性的LB琼脂培养平板上，37℃过夜培养。挑取单菌落，然后提取质粒电泳检测，并在-20℃保存质粒。再利用EcoRI与NotI酶切检测目的片段，之后送菌悬液由公司进行测序，将测序正确的质粒以同样方法转化大肠杆菌实现质粒富集。
(4)pPIC9k-GDH-linker-CBM2转化毕赤酵母宿主菌
接种毕赤酵母GS115单菌落到含有5mL YPD液体培养基的试管中，30℃过夜培养。按1％接种量转接到含有50mL YPD液体培养基的三角瓶，30℃培养过夜，直到OD600＝1.3～1.5；1500g，4℃条件下离心培养液5min，弃上清液，利用50mL冰浴双蒸水重悬细胞；1500g，4℃条件下离心培养液5min，弃上清液，利用25mL冰浴双蒸水重悬细胞；1500g，4℃条件下离心培养液5min，弃上清液，用2mL冰浴的1M的山梨醇溶液重悬细胞；1500g，4℃条件下离心培养液5min，弃上清液，用100μL冰浴的1M的山梨醇溶液重悬细胞，使菌悬液体积大约为150μL；将80μL处理好的感受态细胞和5～20μg经过bglI线性化了的pPIC9k-GDH-linker-CBM2质粒加入一个1.5mL预冷离心管中，混匀。然后把混合液转移入预先冰浴的转化杯中(0.2cm型)；冰浴装有转化混合液的转化杯5min；按照biorad毕赤酵母电转参数设置电转化仪，并启动电脉冲，脉冲后立即往转化杯中加入1mL冰浴的1M的山梨醇溶液，然后把转化液转入一个新的1.5mL离心管中；30℃静置培养2h。吸取GS115转化液100μL涂布MD平板，30℃培养，直到转化子出现。对转化的MD平板上的单克隆进行菌落PCR验证，确保外源基因的整合。
(5)GDH-linker-CBM2融合酶基因诱导表达
接种筛选出来的毕赤酵母重组子于5mL BMGY液体培养基中，30℃，250rpm，振荡培养过夜；取500μL过夜培养物转接于50mL BMGY液体培养基中，30℃，250rpm，振荡培养至OD600＝2～6(对数生长期，大约16-18h)；3000g，离心5min，弃上清，用BMMY液体培养基重悬细胞至OD600＝1.0，甲醇终浓度为1％)置于500mL三角瓶中，用8层灭菌纱布封口，30℃，250rpm，振荡培养5d；8000rpm离心发酵液获得粗酶液，SDS-PAGE检测粗酶液中有无目的蛋白的表达，并进行酶活检测。结果显示在粗酶液中分别以(GGGGS)2、(EAAAK)3、天然linker序列(NL)为连接肽的融合酶都有表达，但酶活存在差异，其中以NL为连接肽 的GDH融合酶发酵液中酶活性最高(2480U/L)，而以(GGGGS)2、(EAAAK)3为连接肽的GDH融合酶发酵液中酶活性分别为1860U/L和1520U/L。
(6)GDH-linker-CBM2融合酶分离纯化
分别将三种连接肽连接的GDH融合酶粗酶液与含有镍的填料混合，于4℃冰箱中转动结合6h。然后用梯度浓度的咪唑溶液洗脱目的蛋白，用3K超滤管、pH 5.0的磷酸氢二钠-柠檬酸缓冲液超滤20mM咪唑洗脱下来的蛋白溶液，4900rpm，4℃，直至流下来的缓冲液的pH为5.0，停止超滤，收集超滤获得的GDH融合酶溶液，进行酶活测定和SDS-PAGE检测目的蛋白。结果显示以(GGGGS)2为连接肽的融合酶可能由于6×His纯化标签无法正常暴露，导致未能获得纯化后的融合酶，而以(EAAAK)3为连接肽的融合酶纯化后酶活性(2360U/L)约为以天然linker序列为连接肽的融合酶活性(92000U/L)的1/4，因此以天然linker序列为连接肽构建的GDH融合酶具有明显酶活性优势，并对其性质进行进一步检测分析。SDS-PAGE检测图谱显示，GDH-NL-CBM2的分子量明显大于野生型GDH的分子量(图1e-1g)，说明CBM2已成功融合到GDH酶分子上，并且由于酶分子的糖基化作用，实际表达酶分子的分子量都要高于预测的分子量。测定融合酶的最适pH为6.0、最适反应温度为50℃，与野生型GDH(pH6.0,37℃)相比，最适pH未发生明显变化，而最适反应温度显著提高，这可能由于CBM2来自于嗜热裂孢菌，具有较高的温度耐受性，融合到酶分子中提高整个酶分子的温度适应性(图2)。
实施例3、GDH和GDH-NL-CBM2融合酶纸基传感元件检测性能对比
将聚二甲基硅氧烷和羊毛脂按10:1的比例混合，60Hz超声混匀30min，打印在制备的孔径为0.1μm的纳米纤维素纸上形成疏水区。在纳米纤维纸亲水区的工作电极打印区滴加实验室前期研究中分离纯化的GDH野生型酶分子(10U，30μL)和25％戊二醛(2μL)、0.5％壳聚糖(8μL)混合溶液以及40μL GDH-NL-CBM2融合酶(10U)，室温干燥4h。再在亲水区分别打印工作电极、对电极和参比电极(图3)。利用电化学工作站三电极体系分别对GDH野生型酶和GDH-NL-CBM2融合酶传感元件性能进行检测。
在电位范围为-0.2V-0.6V时，依次对纸基酶传感元件在pH 7.0的PBS缓冲液和不同浓度的葡萄糖溶液(5mM,10mM，15mM，20mM，25mM，30mM，35mM，40mM，50mM)中进行循环伏安法扫描检测。结果显示两种纸基酶传感元件在PBS缓冲液中都可以检测到一对明显的二茂铁的氧化还原峰，并且氧化峰电位(+0.26V)对应的电流值随着葡萄糖浓度的升高逐渐增加，而还原峰电位(+0.17V)对应的电流值逐渐降低(图4a,4b)，表明两种固定化方式的纸基酶传感元件都具有葡萄糖电催化特性。进一步采用计时电流法对葡萄糖线性 检测范围进行了探究，结果显示在葡萄糖浓度为5mM-50mM范围内，两种纸基酶传感元件电流变化都呈线性相关(R 2≥0.99)(图4c,4d)，但是与GDH酶传感元件检测性能相比(灵敏度10.75μA.mM -1.cm -2，检测限2.62mM)，GDH-NL-CBM2融合酶传感元件具有更高的灵敏度(20.23μA.mM -1.cm -2)和更低检测限2.53mM(S/N＝3)(4e)。同时，与以葡萄糖氧化酶为传感元件的传感器相比，以GDH为传感元件的传感器大大提高对抗坏血酸、尿酸、尿素等物质的抗干扰能力。
GDH-NL-CBM2融合酶纸基传感元件与GDH传感元件相比，具有制作简单，检测性能更加优良等特点，因此，在生物传感器领域应用前景广阔。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种融合酶，其特征在于，所述融合酶包括葡萄糖脱氢酶肽段和碳水化合物结合模块肽段，两部分通过柔性肽链连接。
  2. 如权利要求1所述融合酶，其特征在于，所述葡萄糖脱氢酶肽段来源于Aspergillus niger An76；
    优选的，所述GDH的氨基酸序列如SEQ ID NO:1所示，或与SEQ ID NO:1所示氨基酸序列具有80％及以上相似度、具有同样功能的氨基酸序列也在本发明请求保护的范围之内；
    进一步的，SEQ ID NO:1所示氨基酸序列的编码基因，其序列如SEQ ID NO:2所示；除该基因序列外，还包括由于密码子简并性同样能够翻译得到SEQ ID NO:1所示氨基酸序列的基因序列。
  3. 如权利要求1所述融合酶，其特征在于，所述碳水化合物结合模块肽段选自CBM1、CBM2、CBM3、CBM5、CBM10中的一种。
  4. 如权利要求3所述融合酶，其特征在于，所述碳水化合物结合模块为第2家族CBM，所述CBM2的序列如SEQ ID NO:3所示，或与SEQ ID NO:3所示氨基酸序列具有80％及以上相似度、具有同样功能的氨基酸序列；
    优选的，还提供SEQ ID NO:3所示氨基酸序列的编码基因，其序列如SEQ ID NO:4所示；除该基因序列外，还包括由于密码子简并性同样能够翻译得到SEQ ID NO:3所示氨基酸序列的基因序列。
  5. 如权利要求1所述融合酶，其特征在于，所述柔性肽链为一种连接肽，所述连接肽选自(GGGGS)2、(EAAAK)3、Thermobifida fusca基因组中内切-β-木聚糖酶中连接催化结构域、CBM2结构域的天然linker序列中的一种或任意几种。
  6. 如权利要求5所述融合酶，其特征在于，所述连接肽选自Thermobifida fusca基因组中内切-β-木聚糖酶中连接催化结构域和CBM2结构域的天然linker序列；
    优选的，所述连接肽序列如SEQ ID NO:5所示，或与SEQ ID NO:5所示氨基酸序列具有80％及以上相似度、具有同样功能的氨基酸序列；
    进一步的，还提供SEQ ID NO:5所示氨基酸序列的编码基因，其序列如SEQ ID NO:6所示；除该基因序列外，由于密码子简并性同样能够翻译得到SEQ ID NO:5所示氨基酸序列的基因序列。
  7. 如权利要求1所述融合酶，其特征在于，所述融合酶从N端到C端，其氨基酸序列如SEQ ID NO:11所示；所述融合酶的编码基因如SEQ ID NO:12所示；
    或，所述融合酶氨基酸序列如SEQ ID NO:13或SEQ ID NO:15所示；
    优选的，所述融合酶的制备方法：将SEQ ID NO:12所示编码基因转化到毕赤酵母中，然后进行表达纯化。
  8. 权利要求1-7任一项所述融合酶在制备葡萄糖检测元件中的应用；所述葡萄糖检测元件为纸基生物传感元件。
  9. 一种纸基生物传感元件，其特征在于，所述传感元件为固定权利要求-7任一项所述融合酶的纤维素纸；
    优选的，所述纸基生物传感元件的制备方法如下：将所述融合酶固定到纤维素纸基上、然后在固定有融合酶的纸基上打印电极；
    进一步的，所述制备方法如下：将聚二甲基硅氧烷和羊毛脂按混合超声混匀后打印在纳米纤维素纸上形成疏水区，在纳米纤维纸亲水区的工作电极打印区滴加第一方面所述融合酶，室温干燥，再在亲水区分别打印工作电极、对电极和参比电极。
  10. 一种血糖检测试剂盒，其特征在于，所述试剂盒中包括权利要求1-7任一项所述融合酶的固定化载体和/或权利要求9所述纸基生物传感元件；
    优选的，所述血糖检测试剂盒中，还包括血样采集装置。
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