CN110819642B - A codon-optimized FAD-glucose dehydrogenase gene and its application - Google Patents

Abstract

The invention provides a codon-optimized FAD-glucose dehydrogenase gene, which is derived from Aspergillus niger An76, and the sequence of the gene is shown as SEQ ID No. 2. The original FAD-glucose dehydrogenase gene cannot be successfully expressed in pichia pastoris, can be smoothly expressed in the pichia pastoris through sequence optimization, can obtain purified target protein through separation and purification, and can be used in a glucose biosensor.

Description

Codon-optimized FAD-glucose dehydrogenase gene and application thereof

Technical Field

The invention relates to the technical field of biological enzyme genetic engineering and biosensing, in particular to a codon-optimized FAD-glucose dehydrogenase gene and application thereof in preparation of a biosensing element.

Background

Accurate and rapid monitoring of glucose is of great significance in the industries of medicine, food and the like, and therefore, the biosensor is an ideal method for monitoring glucose due to the characteristics of high specificity, short analysis time, capability of in-situ monitoring, low manufacturing cost and the like. Despite these advantages, there is still a need for biosensors that overcome the problems caused by enzyme molecule sensing elements for detection.

In biosensors using enzymes as sensing elements, oxidoreductases are the main biorecognition elements, among which Glucose Oxidase (GOX) is the most commonly used enzyme that can selectively oxidize glucose to gluconolactone in the presence of oxygen while producing H₂O₂. However, the response of such enzyme biosensors is susceptible to the concentration of oxygen in the measurement medium, whereas the use of artificial electron media is due to O₂The presence of (a) results in a decrease in the glucose detection signal and thus underestimates the glucose content. Furthermore, if it is based on the determination of H₂O₂The levels reflect glucose content and often require very high operating potentials (typically in excess of +600mv compared to standard electrodes) versus H₂O₂Oxidation proceeds and thus many other electroactive compounds present in the biological fluid may also be oxidized causing a false current response. While biosensors based on nicotinamide adenine dinucleotide-dependent glucose dehydrogenase can avoid H₂O₂Is expected to solve the dependence on H₂O₂The problem of detecting glucose, but such enzymes require the additional addition of expensive coenzyme NAD to the reaction system⁺Limiting the use of such enzymes. Therefore, Glucose Dehydrogenase (GDH) having pyrroloquinoline quinone (PQQ) or Flavin Adenine Dinucleotide (FAD) as a coenzyme has been constructed without using O₂As electron acceptors or NAD⁺As suitable candidate enzymes for biosensors of coenzymes, but a part of GDH-PQQ isolated from the outer cytoplasmic membrane requires suitable detergent solubilization, and another part of water-soluble GDH-PQQ has low specificity, which greatly limits the application of such enzymes in biosensors. GDH-FAD (FAD-glucose dehydrogenase gene) has high catalytic efficiency due to its high catalytic activityHigh rate, high substrate specificity, low redox potential and no oxygen₂The characteristic of the electron acceptor makes the construction independent of O₂The glucose sensor has the most potential enzyme molecular element.

GDH-FAD of fungal origin (EC 1.1.99.10) was first discovered in Aspergillus oryzae in 1937, and the presence of GDH-FAD was later identified in Aspergillus terreus, Aspergillus niger, and Aspergillus flavus. However, most GDH-FAD is mainly separated and purified from wild strains at present, and is not easy to be recombined and expressed, which results in that GDH-FAD is used as an enzyme electrode biosensor element, and especially in China, only a small amount of research on GDH-FAD recombination and expression is carried out, so that GDH-FAD high-efficiency recombination and expression and application research in the field of sensors have important significance.

Disclosure of Invention

The invention provides a codon-optimized FAD-glucose dehydrogenase gene.

The codon-optimized FAD-glucose dehydrogenase gene provided by the invention is derived from Aspergillus niger An76, the original gene sequence is shown as SEQ ID No.1, and the sequence-optimized gene sequence is shown as SEQ ID No. 2. The original FAD-glucose dehydrogenase gene cannot be successfully expressed in pichia pastoris, can be smoothly expressed in the pichia pastoris through sequence optimization, and can be separated and purified to obtain a purified target protein.

In another aspect, the present invention also provides a recombinant vector comprising the FAD-glucose dehydrogenase gene. The vector of the invention comprises a cloning vector and an expression vector.

In one embodiment, the cloning vector comprises a vector of the pUC series, e.g., pUC18, pUC 19; the expression vector comprises a vector expressed in escherichia coli and a vector expressed in pichia pastoris, and preferably comprises a vector of pET series, such as pET-21 a; more preferably, the vector for expression in pichia is the pPIC9K vector.

In another aspect, the present invention also provides a recombinant strain comprising the above recombinant vector; the strain comprises escherichia coli or pichia pastoris; preferably, the escherichia coli is escherichia coli DH5 alpha, and the pichia pastoris is pichia pastoris GS 115.

On the other hand, the invention also provides a method for preparing the recombinant FAD-glucose dehydrogenase, which comprises the steps of transforming the sequence-optimized FAD-glucose dehydrogenase gene into pichia pastoris, constructing the pichia pastoris for recombinant expression of the FAD-glucose dehydrogenase, culturing the recombinant pichia pastoris, and purifying the recombinant FAD-glucose dehydrogenase; preferably, the pichia is pichia GS 115.

In another aspect, the present invention also provides an immobilized FAD-glucose dehydrogenase derived from the FAD-glucose dehydrogenase recombinantly expressed by the above-described method.

On the other hand, the invention also provides application of the codon-optimized FAD-glucose dehydrogenase gene in preparation of FAD-glucose dehydrogenase electrodes.

Further, the FAD-glucose dehydrogenase electrode is a FAD-glucose dehydrogenase electrode obtained by immobilizing FAD-glucose dehydrogenase on a modified glassy carbon electrode.

Furthermore, the modified glassy carbon electrode is a glassy carbon electrode modified by adopting ferrocene and a multi-wall carbon nano tube; preferably, the ferrocene is hydroxymethyl ferrocene, and the multi-walled carbon nanotube is a carboxylated multi-walled carbon nanotube

On the other hand, the invention also provides application of the codon-optimized FAD-glucose dehydrogenase gene in preparation of a glucose biosensor.

According to the method, rare codons in an original FAD-glucose dehydrogenase gene are removed according to the difference of the use frequencies of different codons for coding the same amino acid in pichia pastoris, the occurrence of reverse repetitive sequences is avoided, the secondary structure of RNA is guaranteed to be stabilized, splice sites with intron attributes are removed, the FAD-glucose dehydrogenase gene is optimized, the optimized sequence can be successfully expressed in the pichia pastoris, and the purified target protein can be used for preparing FAD-glucose dehydrogenase electrodes.

Drawings

FIG. 1 SDS-PAGE detects codon optimized FAD-glucose dehydrogenase expressed by Pichia pastoris GS115 induced for six consecutive days. M is a protein standard, 1d, 2d, 3d, 4d, 5d and 6d respectively refer to fermentation crude enzyme liquid obtained by induction culture of Pichia pastoris GS115 for 1 day, 2 days, 3 days, 4 days, 5 days and 6 days.

FIG. 2 shows SDS-PAGE detection of codon-optimized FAD-glucose dehydrogenase expressed recombinantly in Pichia pastoris GS115 using imidazole elution at different concentrations, the left graph is a graph showing elution effects using 10mM imidazole, the right graph is a graph showing elution effects using 20mM imidazole, and lanes 1-10 show elution of codon-optimized FAD-glucose dehydrogenase obtained sequentially from 1mL collection tube at a time when recombinant protein is eluted using imidazole at a specific concentration.

FIG. 3 shows electrochemical characterization of recombinant FAD-glucose dehydrogenase electrode.

FIG. 4 shows the response curve of cyclic voltammetry for detecting glucose at different concentrations by using a recombinant FAD-glucose dehydrogenase electrode.

Detailed description of the preferred embodiments

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 codon optimization and cloning of FAD-glucose dehydrogenase Gene

In this example, FAD-glucose dehydrogenase g5086.t1 gene in Aspergillus niger An76 genome was used as An original gene, and the original gene sequence is shown in SEQ ID No. 1. According to the difference of the using frequency of different codons for coding the same amino acid in pichia pastoris, rare codons in an FAD-glucose dehydrogenase g5086.t1 original gene are removed, the occurrence of an inverted repeat sequence is avoided, the secondary structure of RNA is ensured to be stabilized, splice sites of intron attributes are removed, the FAD-glucose dehydrogenase gene is optimized, and according to different strategies, two sequences are optimized and are respectively shown as SEQ ID No.2 and SEQ ID No. 3.

Synthesizing optimized FAD-glucose dehydrogenase gene, and utilizing Nanjing Novozam according to principle of homologous recombination

The product of Ultra One Step Cloning Kit homologous recombines the codon optimized FAD-glucose dehydrogenase gene onto pPIC9K plasmid vector in the form of 10. mu.L (1. mu.L pPIC9K vector linearized with EcoRI and NotI, 2. mu.L PCR fragment of FAD-glucose dehydrogenase gene, 5. mu.L 2 XClonexpress Mix, 2. mu.L ddH)₂O), sucking, pumping, uniformly mixing, reacting for 15min at 50 ℃, immediately placing on ice, and cooling to obtain a product of homologous recombination of the FAD-glucose dehydrogenase gene and the pPIC9K plasmid vector.

Example 2 transformation of Escherichia coli-enriched plasmid with FAD-glucose dehydrogenase Gene

Respectively mixing the original FAD-glucose dehydrogenase gene (SEQ ID No.1) and the codon-optimized FAD-glucose dehydrogenase gene (SEQ ID No.2 and SEQ ID No.3) and pPIC9K homologous recombinant products with E.coli DH5 alpha, thermally shocking for 90s, spreading on 100ug/mL ampicillin-resistant LB agar culture plates, and culturing overnight at 37 ℃. Single colonies were picked, followed by plasmid electrophoresis detection and plasmid preservation at-20 ℃. And carrying out enzyme digestion detection on the target fragment by utilizing EcoRI and NotI, then sending the target fragment to a bacterial suspension for sequencing by a company, and converting the plasmid with correct sequencing into escherichia coli by the same method to realize plasmid enrichment.

Example 3 transformation of Pichia host bacteria with FAD-glucose dehydrogenase Gene

Inoculating Pichia pastoris GS115 single colony into a test tube containing 5mLYPD liquid culture medium, and culturing at 30 ℃ overnight. Inoculating into a triangular flask containing 50mL of YPD liquid culture medium according to the inoculation amount of 1%, and culturing overnight at 30 ℃ until OD600 is 1.3-1.5; 1500g, centrifuging the culture solution for 5min at 4 ℃, discarding the supernatant, and resuspending the cells by using 50mL ice bath double distilled water; centrifuging the culture solution at 4 ℃ for 5min at 1500g, removing the supernatant, and resuspending the cells by using 25mL ice-bath double distilled water; 1500g, centrifuging the culture solution at 4 ℃ for 5min, discarding the supernatant, and resuspending the cells with 2mL of ice-bath 1M sorbitol solution; 1500g, centrifuging the culture solution at 4 ℃ for 5min, discarding the supernatant, and resuspending the cells with 1mL of ice-bath 1M sorbitol solution to make the volume of the bacterial suspension about 1.5 mL; add 80. mu.L of treated competent cells and 5-20. mu.g of SacI-linearized plasmid containing recombinant FAD-glucose dehydrogenase obtained in example 1-2 into a 1.5mL precooling centrifuge tube, and mix well. Then the mixture was transferred into a transformation cup (type 0.2 cm) pre-iced; ice-bath the transformation cup containing transformation mixture for 5 min; an electric converter (Voltage (V): 2000; Capacitance (muF): 25; Resistance (omega): 200; Cuvette (mm):2) was set according to the electric conversion parameters of the Pichia Biorad, and an electric pulse was started, 1mL of ice-bath 1M sorbitol solution was added to the conversion cup immediately after the pulse, and then the conversion solution was transferred to a new 1.5mL centrifuge tube; standing and culturing for 2h at 30 ℃. mu.L of GS115 transformation solution was aspirated to coat MD plates and cultured at 30 ℃ until transformants appeared. Colony PCR verification is carried out on the monoclone on the transformed MD plate, and the integration of the exogenous gene is ensured.

Example 4 Induction of expression of FAD-glucose dehydrogenase Gene

Inoculating the screened pichia pastoris recombinant into 5mL of BMGY liquid medium, performing shaking culture at 30 ℃ and 250rpm overnight; 500 μ L of overnight culture was transferred to 50mL BMGY liquid medium at 30 ℃, 250rpm, and shake-cultured until OD600 was 2-6 (logarithmic growth phase, approximately 16-18 h); 3000g, centrifuge for 5min, discard the supernatant, resuspend the cells with BMMY broth to OD600 ═ 1.0, final methanol concentration of 1%; 4) placing in a 500mL triangular flask, sealing with 8 layers of sterilized gauze, culturing at 30 deg.C and 250rpm under continuous shaking for 6 days, and sampling 1mL per day; SDS-PAGE detects whether the foreign gene is expressed or not; the results show that the original FAD-glucose dehydrogenase gene (SEQ ID No.1) can not be successfully expressed in Pichia pastoris, the codon-optimized FAD-glucose dehydrogenase gene (SEQ ID No.2) can be successfully expressed in Pichia pastoris (FIG. 1), but the codon-optimized FAD-glucose dehydrogenase gene shown in SEQ ID No.3 can not be successfully expressed in Pichia pastoris; this is probably because the FAD-glucose dehydrogenase gene obtained by this codon optimization method cannot be expressed in pichia pastoris expression due to the influence of factors other than codons, and although the original gene sequence can be optimized according to codon preference, this is only a theoretical possibility, and it is closely related to various factors whether the gene can be successfully expressed in pichia pastoris or not in actual operation.

Example 5 isolation and purification of codon-optimized FAD-glucose dehydrogenase

Mixing the crude enzyme solution with target protein expression detected by SDS-PAGE with filler containing nickel, and rotating and combining for 6h in a refrigerator at 4 ℃. Then, the hetero-protein was eluted with an imidazole solution at a concentration of 5mM, the FAD-glucose dehydrogenase of interest was eluted with 10mM and 20mM imidazole solutions, and the protein of interest was detected by SDS-PAGE (FIG. 2). Ultrafiltering the protein solution eluted with 10mM and 20mM imidazole in disodium hydrogen phosphate-citric acid buffer solution of pH 5.0 at 4900rpm at 4 deg.C until the pH of the buffer solution flowing down is 5.0, stopping the ultrafiltration, and collecting the enzyme solution obtained by ultrafiltration with a 3K ultrafiltration tube. FIG. 2 shows the electrophoretogram of recombinant proteins eluted with imidazole of different concentrations after the successful expression of the codon-optimized FAD-glucose dehydrogenase gene (SEQ ID No.2) in Pichia pastoris, and it can be seen from FIG. 2 that the elution with 10mM and 20mM imidazole can yield purified recombinant FAD-glucose dehydrogenase (0.48 mg/mL).

Example 6 codon optimized immobilization of FAD-glucose dehydrogenase

Pretreating a glassy carbon electrode: polishing glassy carbon electrode firstly, the glassy carbon electrode is cleaned by deionized water and then is sequentially coated with Al with phi of 0.05 mu m and 50 mu m₂O₃Polishing the polishing powder until the surface of the electrode is mirror-finished, rinsing the electrode with distilled water, ultrasonically cleaning the electrode for 1min, taking out the electrode, naturally drying the electrode, ultrasonically cleaning the electrode for 1min by using nitric acid (V: V is 1:1) and ethanol (V: V is 1:1) sequentially, taking out the electrode, rinsing the electrode with distilled water, and naturally drying the electrode for later use.

Cyclic voltammetric scanning in potassium ferricyanide solution: 0.0329g of potassium ferricyanide and 2.022g of KNO were weighed out₃Placing in a beaker, adding 80mL of distilled water, stirring to dissolve, transferring to a 100mL volumetric flask, shaking to constant volume, and making into 1.0 × 10^-3mol/L potassium ferricyanide solution (containing 0.2mol/L KNO)₃) And scanning the glassy carbon electrode in the prepared potassium ferricyanide solution by cyclic voltammetryThe potential difference is within 80mV and approaches 64 mV.

Modifying a glassy carbon electrode by using a carboxylated multi-wall carbon nanotube: 0.3g of carboxylated multi-walled carbon nanotubes was weighed and placed in a 50mL beaker, 0.5g of monoethyl 3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.5g N-hydroxysuccinimide (NHS) were added and dissolved in 10mL of distilled water, and the carbon nanotubes were activated by standing at room temperature for 6 h. Centrifuging at 13000 Xg for 10min, removing supernatant, taking out the precipitated carbon tube, redissolving with appropriate amount of distilled water, repeating the centrifugation step, adding distilled water to wash the carbon tube until the carbon tube is neutral, and drying for later use. 0.3g of the prepared carbon tube is weighed and dissolved in 100mL of distilled water by ultrasonic wave to prepare a carboxylated multi-walled carbon nanotube solution with the concentration of 3g/L, 7uL of the carboxylated multi-walled carbon nanotube is dripped on the surface of the glassy carbon electrode, and the glassy carbon electrode is naturally aired for later use.

The hydroxymethyl ferrocene-multiwalled carbon nanotube modified glassy carbon electrode comprises the following steps: and (3) in an environment of 4 ℃, soaking the glassy carbon electrode modified by the carboxylated multi-walled carbon nano tube in 1g/L hydroxymethyl ferrocene solution for 24h, and storing in a refrigerator for later use.

Cross-linking FAD-glucose dehydrogenase with cross-linker: 0.5g of chitosan is weighed and dissolved in 100mL of distilled water, and acetic acid is dropwise added while stirring until the chitosan is completely dissolved, so as to prepare a 0.5% chitosan solution. The FAD-glucose dehydrogenase (1mg/mL) purified in example 5, a 0.5% chitosan solution and a 25% glutaraldehyde solution were mixed at equal volume ratios to obtain a cross-linked enzyme solution containing FAD-glucose dehydrogenase, chitosan and glutaraldehyde, and the obtained cross-linked enzyme solution was stored at 4 ℃ for use.

Immobilization of FAD-glucose dehydrogenase: and taking out the glassy carbon electrode modified by the hydroxymethyl ferrocene-multi-walled carbon nano tube, washing with distilled water, and airing. And (3) dropwise adding 7 mu L of prepared crosslinking enzyme solution, airing at 4 ℃, then leaching with phosphate buffer solution with pH of 7.0 to obtain the FAD-glucose dehydrogenase electrode, and storing at 4 ℃ for later use.

Example 7 electrochemical characterization of FAD-glucose dehydrogenase electrode

A three-electrode system is adopted to detect and explore the electrochemical properties of an enzyme electrode, when the potential range is-0.8-0.8V, the hydroxymethyl ferrocene-multi-walled carbon nanotube modified glassy carbon electrode is scanned and detected in double distilled water and 1mM Glucose in sequence and in FAD-Glucose dehydrogenase-chitosan-hydroxymethyl ferrocene-multi-walled carbon nanotube modified glassy carbon electrode is scanned and detected in 1mM Glucose by a cyclic voltammetry method, the result shows that when the glassy carbon electrode only modifies the hydroxymethyl ferrocene-multi-walled carbon nanotube without modifying FAD-Glucose dehydrogenase, no redox peak exists in water or Glucose solution, and when the modified FAD-Glucose dehydrogenase is detected in the Glucose solution, obvious redox peaks appear at the potentials of +0.4 and +0.2, which indicates that the FAD-Glucose dehydrogenase electrode can realize the detection of Glucose, and finally, performing cyclic voltammetry scanning on glucose solutions with different concentrations (3.8mmol.L-1, 5.8mmol.L-1, 6.3mmol.L-1, 6.9mmol.L-1, 7.2mmol.L-1, 8.0mmol.L-1) by using the glassy carbon electrode modified by the FAD-glucose dehydrogenase-chitosan-hydroxymethyl ferrocene-multi-walled carbon nanotube, wherein the result is shown in FIG. 4, and the current value corresponding to the oxidation peak is gradually increased along with the increase of the sugar concentration, which indicates that the prepared FAD-glucose dehydrogenase electrode can be applied to a glucose biosensor as a sensing element to realize the quantitative detection of glucose in a sample.

Sequence listing

<120> codon-optimized FAD-glucose dehydrogenase gene and application thereof

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ccattccagc tgtgtggcca ccttacctct actttgtacg ctgttgccga gagagcttca 1680

gacttgatca aggaatccta tcatcaccac caccatcatt aa 1722

Claims (11)

Translated fromChinese

1.一种密码子优化的FAD-葡萄糖脱氢酶基因，其特征在于，所述基因的核苷酸序列如SEQ ID No.2所示。1. A codon-optimized FAD-glucose dehydrogenase gene, wherein the nucleotide sequence of the gene is shown in SEQ ID No.2.2.包含权利要求1所述基因的重组载体，所述载体骨架为pPIC9K载体。2. A recombinant vector comprising the gene of claim 1, wherein the vector backbone is a pPIC9K vector.3.包含权利要求2所述重组载体的重组菌株。3. A recombinant strain comprising the recombinant vector of claim 2.4.根据权利要求3所述的重组菌株，其特征在于，所述菌株包括大肠杆菌或毕赤酵母。4. The recombinant strain according to claim 3, wherein the strain comprises Escherichia coli or Pichia pastoris.5.根据权利要求4所述的重组菌株，其特征在于，所述大肠杆菌为大肠杆菌DH5α。5. The recombinant strain according to claim 4, wherein the Escherichia coli is Escherichia coli DH5α.6.根据权利要求4所述的重组菌株，其特征在于，所述毕赤酵母为毕赤酵母GS115。6. The recombinant strain according to claim 4, wherein the Pichia pastoris is Pichia pastoris GS115.7.一种制备重组FAD-葡萄糖脱氢酶的方法，其特征在于，所述方法包括如下步骤：7. a method for preparing recombinant FAD-glucose dehydrogenase, is characterized in that, described method comprises the steps:将权利要求1所述的基因转化到毕赤酵母中，构建重组表达FAD-葡萄糖脱氢酶的毕赤酵母，然后，对重组的毕赤酵母进行培养，并对重组的FAD-葡萄糖脱氢酶进行纯化；所述毕赤酵母为毕赤酵母GS115。Transforming the gene of claim 1 into Pichia pastoris, constructing Pichia pastoris expressing FAD-glucose dehydrogenase recombinantly, then culturing the recombinant Pichia pastoris, and culturing the recombinant FAD-glucose dehydrogenase Purified; the Pichia pastoris was Pichia pastoris GS115.8.权利要求1所述的基因在制备FAD-葡萄糖脱氢酶电极中的应用。8. The application of the gene of claim 1 in the preparation of FAD-glucose dehydrogenase electrode.9.根据权利要求8所述的应用，其特征在于，所述FAD-葡萄糖脱氢酶电极为将FAD-葡萄糖脱氢酶固定在修饰的玻碳电极上得到的葡萄糖脱氢酶电极。9 . The application according to claim 8 , wherein the FAD-glucose dehydrogenase electrode is a glucose dehydrogenase electrode obtained by immobilizing FAD-glucose dehydrogenase on a modified glassy carbon electrode. 10 .10.根据权利要求9所示的应用，其特征在于，所述修饰的玻碳电极为采用二茂铁和多壁碳纳米管修饰的玻碳电极。10 . The application according to claim 9 , wherein the modified glassy carbon electrode is a glassy carbon electrode modified with ferrocene and multi-walled carbon nanotubes. 11 .11.权利要求1所述的基因在制备葡萄糖生物传感器中的应用。11. Use of the gene of claim 1 in the preparation of a glucose biosensor.

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