WO2021104537A1 - Codon-optimized fad-glucose dehydrogenase gene and application thereof - Google Patents

Abstract

Provided is a codon-optimized FAD-glucose dehydrogenase gene, derived from Aspergillus niger An76, the sequence thereof being as shown in SEQ ID No. 2. An initial FAD-glucose dehydrogenase gene cannot be successfully expressed in Pichia pastoris, but upon sequence optimization, can be successfully expressed in Pichia pastoris. In addition, a purified target protein can be obtained by means of separation and purification, and the recombinant FAD-glucose dehydrogenase may be used in glucose biosensors.

Description

A codon-optimized FAD-glucose dehydrogenase gene and its application Technical field

The invention relates to the fields of biological enzyme genetic engineering and biosensing technology, in particular to a codon-optimized FAD-glucose dehydrogenase gene and its application in preparing biosensing elements.

Background technique

Accurate and rapid glucose monitoring is of great significance in the pharmaceutical, food and other industries. For this reason, biosensors have become an ideal method for glucose monitoring due to their high specificity, short analysis time, in-situ monitoring, and low manufacturing cost. Despite these advantages, biosensors still need to overcome the problems caused by enzyme molecular sensing elements for detection.

In biosensors using enzymes as the sensing element, oxidoreductase is the main biorecognition element. Among them, glucose oxidase (GOX) is the most commonly used enzyme. GOX can selectively oxidize glucose into the presence of oxygen. Glucose lactone, while producing H ₂ O ₂ . However, the response of this type of enzyme biosensor is easily affected by the oxygen concentration in the measurement medium, and the use of artificial electronic media will _{reduce the glucose detection signal due to the presence of O 2} and thus underestimate the glucose content. In addition, if it is based on measuring the H ₂ O ₂ level to reflect the glucose content, a very high working potential (compared with standard electrodes, usually more than +600mv) is often required _{to oxidize H 2} O ₂ , so it is present in biological liquids. Many other electroactive compounds may also be oxidized to cause a false current response. The biosensor based on nicotinamide adenine dinucleotide-dependent glucose dehydrogenase can avoid the production of H ₂ O ₂ and is expected to solve the problem of relying on H ₂ O _{2 to} detect glucose, but this type of enzyme must be added to the reaction system. The addition of expensive coenzyme NAD ⁺ limits the application of this type of enzyme. Therefore, glucose dehydrogenase (GDH) with pyrroloquinoline quinone (PQQ) or flavin adenine dinucleotide (FAD) as a coenzyme has become _{a biosensor that does not use O 2} as an electron acceptor or NAD ⁺ as a coenzyme. Suitable candidate enzymes, but part of GDH-PQQ isolated from the outer cytoplasmic membrane needs to be dissolved by a suitable detergent, while another part of the water-soluble GDH-PQQ has very low specificity, which greatly limits the use of this enzyme in biosensors. application. GDH-FAD (FAD-Glucose Dehydrogenase Gene) has the characteristics of high catalytic efficiency, strong substrate specificity, low redox potential and no O ₂ as the electron acceptor, making it a construction independent of O ₂ The most promising enzyme molecular component of glucose sensor.

Fungal-derived GDH-FAD (EC 1.1.99.10) was first discovered in Aspergillus oryzae in 1937, and the existence of GDH-FAD was also identified in Aspergillus terreus, Aspergillus niger, and Aspergillus flavus. However, most of GDH-FAD is mainly isolated and purified from wild strains, and it is not easy to express recombinantly. This also leads to less research on GDH-FAD as enzyme electrode biosensing element, especially in China, there are only a few about GDH-FAD recombination The study of expression, therefore, the high-efficiency recombinant expression of GDH-FAD and its application in the field of sensors are of great significance.

Summary of the invention

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

The codon-optimized FAD-glucose dehydrogenase gene provided by the present invention is derived from Aspergillus niger An76, and its original gene sequence is shown in SEQ ID No. 1, and the sequence-optimized gene sequence is shown in SEQ ID No. 2. The original FAD-glucose dehydrogenase gene cannot be successfully expressed in Pichia pastoris. After sequence optimization, it can be successfully expressed in Pichia pastoris, and after isolation and purification, a purified target protein can be obtained.

On the other hand, the present invention also provides a recombinant vector containing the FAD-glucose dehydrogenase gene. The vectors of the present invention include cloning vectors and expression vectors.

In one embodiment, the cloning vector includes the pUC series of vectors, for example, pUC18, pUC19; the expression vector includes the vector expressed in E. coli and the vector expressed in Pichia pastoris, preferably, includes the pET series The vector, such as pET-21a; more preferably, the vector expressed in Pichia pastoris is the pPIC9K vector.

On the other hand, the present invention also provides a recombinant strain containing the above-mentioned recombinant vector; the strain includes Escherichia coli or Pichia pastoris; preferably, the Escherichia coli is Escherichia coli DH5α, and the Pichia pastoris is Pichia pastoris GS115 .

On the other hand, the present invention also provides a method for preparing recombinant FAD-glucose dehydrogenase, which comprises transforming the sequence-optimized FAD-glucose dehydrogenase gene into Pichia pastoris to construct recombinant expression FAD- Pichia pastoris for glucose dehydrogenase, then, the recombinant Pichia pastoris is cultured, and the recombinant FAD-glucose dehydrogenase is purified; preferably, the Pichia pastoris is Pichia pastoris GS115.

On the other hand, the present invention also provides an immobilized FAD-glucose dehydrogenase, which is derived from the FAD-glucose dehydrogenase recombinantly expressed by the above method.

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

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

Further, the modified glassy carbon electrode is a glassy carbon electrode modified with ferrocene and multi-walled carbon nanotubes; preferably, the ferrocene is hydroxymethyl ferrocene, and the multi-walled carbon nanotubes Carboxylated multi-walled carbon nanotubes

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

The present invention removes rare codons in the original FAD-glucose dehydrogenase gene based on the difference in the frequency of use of different codons encoding the same amino acid in Pichia pastoris, avoids the appearance of inverted repeats, and also ensures stable RNA secondary Structure, remove splicing sites with intron properties, optimize FAD-glucose dehydrogenase gene, the optimized sequence can be successfully expressed in Pichia pastoris, and the purified target protein can be used to prepare FAD-glucose dehydrogenase electrode.

Description of the drawings

Figure 1. SDS-PAGE detection of codon-optimized FAD-glucose dehydrogenase induced by Pichia pastoris GS115 for six consecutive days. M is a protein standard, and 1d, 2d, 3d, 4d, 5d, 6d refer to the fermentation crude enzyme solution obtained when Pichia pastoris GS115 is induced to culture for 1, 2, 3, 4, 5, and 6 days, respectively.

Figure 2. SDS-PAGE detection of the use of different concentrations of imidazole eluted in Pichia pastoris GS115 recombinant expression of codon-optimized FAD-glucose dehydrogenase, the left picture is the elution effect of 10mM imidazole, the right picture is the use of 20mM The elution effect diagram of imidazole, lanes 1-10 are the codon-optimized FAD-glucose dehydrogenase eluate obtained in sequence when the recombinant protein is eluted with a specific concentration of imidazole. Each 1 mL is 1 collection tube.

Figure 3. Electrochemical characterization diagram of recombinant FAD-glucose dehydrogenase electrode.

Figure 4. Recombinant FAD-glucose dehydrogenase electrode detects the cyclic voltammetry response curve of different concentrations of glucose.

Implementation

The present invention will be further explained below in conjunction with the embodiments. The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Anyone familiar with the profession may use the technical content disclosed above. Change to an equivalent embodiment with the same change. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the solution of the present invention falls within the protection scope of the present invention.

Example 1. Codon optimization and cloning of FAD-glucose dehydrogenase gene

In this embodiment, the FAD-glucose dehydrogenase g5086.t1 gene in the genome of Aspergillus niger An76 is the original gene, and the original gene sequence is shown in SEQ ID No. 1. According to the difference in the frequency of use of different codons encoding the same amino acid in Pichia pastoris, the rare codons in the original gene of FAD-glucose dehydrogenase g5086.t1 are removed, to avoid the appearance of inverted repeats, and to ensure stable RNA two Hierarchical structure, removing splicing sites with intron properties, optimizing the FAD-glucose dehydrogenase gene, and optimizing two sequences according to different strategies, as shown in SEQ ID No. 2 and SEQ ID No. 3, respectively.

Synthesize the optimized FAD-glucose dehydrogenase gene and use Nanjing Novozan according to the principle of homologous recombination

The Ultra One Step Cloning Kit product homologous recombination of codon-optimized FAD-glucose dehydrogenase gene into pPIC9K plasmid vector, the reaction system is 10μL (1μL pPIC9K vector linearized with EcoRI and NotI, 2μL FAD-glucose dehydrogenase Gene PCR fragment, 5μL 2×ClonExpress Mix, 2μL ddH ₂ O), pipet and mix well, react at 50°C for 15 minutes, and immediately place it on ice to cool down to obtain FAD-glucose dehydrogenase gene homologous recombination with pPIC9K plasmid vector product.

Example 2. FAD-Glucose Dehydrogenase Gene Transformation of Escherichia coli Enriched Plasmid

The original FAD-glucose dehydrogenase gene (SEQ ID No. 1) and the codon-optimized FAD-glucose dehydrogenase gene (SEQ ID No. 2, SEQ ID No. 3) and the homologous recombination product of pPIC9K were respectively combined with E.coli DH5α was mixed, heat shocked for 90s, spread on a 100ug/mL ampicillin-resistant LB agar culture plate, and cultured overnight at 37°C. Pick a single colony, then extract the plasmid for electrophoresis detection, and store the plasmid at -20°C. Then use EcoRI and NotI to digest the target fragments, and then send the bacterial suspension to the company for sequencing. The plasmids with the correct sequence are transformed into E. coli in the same way to achieve plasmid enrichment.

Example 3 Transformation of Pichia pastoris host bacteria with FAD-glucose dehydrogenase gene

Inoculate a single colony of Pichia pastoris GS115 into a test tube containing 5mLYPD liquid medium, and cultivate overnight at 30°C. Transfer to an Erlenmeyer flask containing 50mL YPD liquid medium according to 1% inoculum, and incubate at 30°C overnight until OD600=1.3～1.5; 1500g, centrifuge the culture solution at 4°C for 5min, discard the supernatant, and use a 50mL ice bath Resuspend the cells in double-distilled water; centrifuge the culture solution at 1500g for 5 minutes at 4°C, discard the supernatant, and resuspend the cells in 25 mL of ice-bath double-distilled water; centrifuge the culture solution at 1500g for 5 minutes at 4°C, discard the supernatant, Resuspend the cells with 2mL ice-bath 1M sorbitol solution; centrifuge the culture solution at 1500g for 5min at 4℃, discard the supernatant, and resuspend the cells with 1mL ice-bath 1M sorbitol solution to make the volume of the bacterial suspension approximately 1.5mL; 80μL of processed competent cells and 5～20μg of the recombinant FAD-glucose dehydrogenase plasmid obtained in Example 1-2 linearized by SacI were added to a 1.5mL pre-cooled centrifuge tube and mixed uniform. Then transfer the mixture into a pre-ice-bathed transformation cup (0.2cm type); the ice-bath is filled with the transformation cup for the conversion mixture for 5 minutes; set the electrotransformer according to the Biorad Pichia electrotransformation parameters (Voltage(V): 2000; Capacitance(μF): 25; Resistance(Ω): 200; Cuvette(mm): 2), and start the electric pulse. Immediately after the pulse, add 1 mL of 1M sorbitol solution in an ice bath to the transformation cup, and then transfer the transformation solution to Put it into a new 1.5mL centrifuge tube; incubate at 30°C for 2h. Aspirate 100 μL of GS115 transformation solution to spread on MD plates and incubate at 30°C until transformants appear. Perform colony PCR verification on the single clones on the transformed MD plates to ensure the integration of exogenous genes.

Example 4 Induced expression of FAD-glucose dehydrogenase gene

Inoculate the selected Pichia pastoris recombinants in 5mL BMGY liquid medium, 30℃, 250rpm, shaking culture overnight; transfer 500μL of overnight culture to 50mL BMGY liquid medium, 30℃, 250rpm, shaking culture to OD600 ＝2～6 (logarithmic growth phase, about 16-18h); 3000g, centrifuge for 5min, discard the supernatant, resuspend the cells in BMMY liquid medium to OD600 = 1.0, the final concentration of methanol is 1%; 4) Place in 500mL In the Erlenmeyer flask, seal with 8 layers of sterile gauze, 30℃, 250rpm, continuous shaking culture for 6 days, sampling 1mL every day; SDS-PAGE detects the expression of foreign genes; the results show that the original FAD-glucose dehydrogenase The gene (SEQ ID No.1) cannot be successfully expressed in Pichia pastoris, and the codon-optimized FAD-glucose dehydrogenase gene (SEQ ID No.2) can be successfully expressed in Pichia pastoris (Figure 1), but , The codon-optimized FAD-glucose dehydrogenase gene shown in SEQ ID No. 3 cannot be successfully expressed in Pichia pastoris; this may be because the FAD-glucose dehydrogenase gene obtained by this codon optimization method is in The expression of Pichia pastoris is affected by factors other than codons, which makes it impossible to express. Although the original gene sequence can be optimized according to the codon preference, this is only a theoretical possibility. The smooth expression in red yeast is closely related to many factors.

Example 5. Separation and purification of codon-optimized FAD-glucose dehydrogenase

Mix the crude enzyme solution with the expression of the target protein detected by SDS-PAGE with the filler containing nickel, and rotate and combine in a refrigerator at 4°C for 6 hours. Then the contaminated protein was eluted with 5mM imidazole solution, the target FAD-glucose dehydrogenase was eluted with 10mM and 20mM imidazole solution, and the target protein was detected by SDS-PAGE (Figure 2). Ultrafiltration of the protein solution eluted with 10mM and 20mM imidazole with pH 5.0 disodium hydrogen phosphate-citrate buffer, 4900rpm, 4℃, until the pH of the flowing buffer is 5.0, stop ultrafiltration, use 3K ultrafiltration The tube collects the enzyme solution obtained by ultrafiltration. Figure 2 shows the electrophoresis of the recombinant protein eluted with different concentrations of imidazole after the codon-optimized FAD-glucose dehydrogenase gene (SEQ ID No. 2) was successfully expressed in Pichia pastoris, as shown in Figure 2. It can be seen that the purified recombinant FAD-glucose dehydrogenase (0.48 mg/mL) can be obtained by eluting with 10 mM and 20 mM imidazole.

Example 6. Immobilization of codon-optimized FAD-glucose dehydrogenase

Pretreatment of the glassy carbon electrode: Polish the glassy carbon electrode first, wash the glassy carbon electrode with deionized water, and then use φ=0.05μm and 50μm Al ₂ O ₃ polishing powder to polish until the mirror surface, rinse with distilled water Ultrasonic cleaning for 1 min, take it out and let it dry naturally, then use nitric acid (V:V=1:1) and ethanol (V:V=1:1) to ultrasonically clean it for 1 min. Take it out and rinse the electrode surface with distilled water and dry it naturally for later use.

Cyclic voltammetry scan of potassium ferricyanide solution: Weigh 0.0329g potassium ferricyanide and 2.022g KNO ₃ in a beaker, add 80mL distilled water and stir to dissolve it, then transfer to a 100mL volumetric flask to constant volume and shake to make it 1.0×10 ^-3 mol/L potassium ferricyanide solution (containing 0.2mol/L KNO ₃ ), and the glassy carbon electrode was scanned by cyclic voltammetry in the prepared potassium ferricyanide solution. The potential difference is Within 80mV and close to 64mV.

Carboxylated multi-walled carbon nanotubes modified glassy carbon electrode: Weigh 0.3g of carboxylated multi-walled carbon nanotubes in a 50mL beaker, add 0.5g of hydrochloric acid monoethyl-3-(3-dimethylaminopropyl) ) Carbodiimide (EDC) and 0.5 g N-hydroxysuccinimide (NHS), and dissolve the three in 10 mL of distilled water, and let the carbon tube be activated at room temperature for 6 hours. Centrifuge at 13000×g for 10 minutes, discard the supernatant, take out the precipitated carbon tube, reconstitute it with an appropriate amount of distilled water, repeat this step of centrifugation and add distilled water to wash until the carbon tube is washed to neutrality, and then dry for use. Weigh 0.3 g of the carbon tube prepared above and dissolve it in 100 mL of distilled water ultrasonically to prepare a solution of carboxylated multi-walled carbon nanotubes with a concentration of 3 g/L. Take 7 uL of carboxylated multi-walled carbon nanotubes and apply drops on the surface of the glassy carbon electrode. Let it dry naturally and set aside.

Hydroxymethylferrocene-multiwall carbon nanotube modified glassy carbon electrode: Place the carboxylated multiwall carbon nanotube modified glassy carbon electrode in a 1g/L hydroxymethylferrocene solution at 4℃ Soak in medium for 24h and keep in refrigerator for later use.

Cross-linking of FAD-glucose dehydrogenase and cross-linking agent: Weigh 0.5 g of chitosan and dissolve it in 100 mL of distilled water, add acetic acid dropwise while stirring until all the chitosan is dissolved to prepare a 0.5% chitosan solution. The FAD-glucose dehydrogenase (1 mg/mL) purified in Example 5, 0.5% chitosan solution, and 25% glutaraldehyde solution were mixed in equal proportions by volume to obtain FAD-glucose dehydrogenase and chitosan , Glutaraldehyde cross-linking enzyme solution, and the prepared cross-linking enzyme solution is stored at 4 ℃ for use.

Immobilization of FAD-glucose dehydrogenase: Take out the hydroxymethylferrocene-multiwall carbon nanotube modified glassy carbon electrode, wash it with distilled water, and dry it. Add 7 μL of the prepared cross-linking enzyme solution dropwise to dry at 4°C, and then rinse with a phosphate buffer solution of pH 7.0 to prepare an FAD-glucose dehydrogenase electrode, which is stored at 4°C for future use.

Example 7. Electrochemical characterization of FAD-glucose dehydrogenase electrode

A three-electrode system was used to detect and explore the electrochemical properties of the enzyme electrode. When the potential range was -0.8-0.8V, the hydroxymethylferrocene-multi-walled carbon nanotube modified glassy carbon electrode was used in double distilled water. , 1mM Glucose and FAD-glucose dehydrogenase-chitosan-hydroxymethylferrocene-multi-walled carbon nanotube modified glassy carbon electrode was scanned and detected by cyclic voltammetry in 1mM Glucose, the results showed that When the glassy carbon electrode is only modified with hydroxymethylferrocene-multi-walled carbon nanotubes without modifying FAD-glucose dehydrogenase, there is no redox peak in water or glucose solution, and FAD-glucose dehydrogenation is modified When the enzyme is detected in the glucose solution later, obvious redox peaks appear at the potentials of +0.4 and +0.2, indicating that the FAD-glucose dehydrogenase electrode can realize the detection of glucose, and finally use FAD-glucose dehydrogenase-chitopolymer Sugar-hydroxymethylferrocene-multi-walled carbon nanotube modified glassy carbon electrode for different concentrations of glucose solutions (3.8mmol.L-1, 5.8mmol.L-1, 6.3mmol.L-1, 6.9mmol. L-1, 7.2mmol.L-1, 8.0mmol.L-1) was scanned by cyclic voltammetry. The results are shown in Figure 4. As the sugar concentration increases, the current value corresponding to the oxidation peak gradually increases, indicating that the obtained The FAD-glucose dehydrogenase electrode can be used as a sensing element in a glucose biosensor to achieve quantitative detection of glucose in a sample.

Claims (10)

1. A codon-optimized FAD-glucose dehydrogenase gene is characterized in that the nucleotide sequence of the gene is shown in SEQ ID No.2.
2. A recombinant vector containing the gene of claim 1.
3. The recombinant vector of claim 2, wherein the vector backbone is derived from a vector that can be expressed in Pichia pastoris.
4. The recombinant vector of claim 3, wherein the vector backbone is a pPIC9K vector.
5. A recombinant strain comprising the recombinant vector of any one of claims 2-4.
6. The recombinant strain according to claim 5, wherein the strain comprises Escherichia coli or Pichia pastoris; preferably, the Escherichia coli is Escherichia coli DH5α; preferably, the Pichia pastoris is Pichia pastoris GS115 .
7. A method for preparing recombinant FAD-glucose dehydrogenase, characterized in that the method comprises the following steps:

   Transform the gene of claim 1 into Pichia pastoris to construct a Pichia pastoris that recombinantly express FAD-glucose dehydrogenase, then culture the recombinant Pichia pastoris, and analyze the recombinant FAD-glucose dehydrogenase Purify;

   Preferably, the Pichia pastoris is Pichia pastoris GS115.
8. The use of the gene of claim 1 in the preparation of FAD-glucose dehydrogenase electrodes.
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; preferably, the The modified glassy carbon electrode is a glassy carbon electrode modified with ferrocene and multi-wall carbon nanotubes.
10. The use of the gene of claim 1 in the preparation of a glucose biosensor.

Images