CN115747182A - Deglycosylated cellobiose dehydrogenase, lactose biosensor and application of deglycosylated cellobiose dehydrogenase and lactose biosensor - Google Patents
Deglycosylated cellobiose dehydrogenase, lactose biosensor and application of deglycosylated cellobiose dehydrogenase and lactose biosensor Download PDFInfo
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Abstract
The invention relates to the technical field of biological enzyme genetic engineering and biosensing, in particular to a deglycosylated cellobiose dehydrogenase, a lactose biosensor and application thereof. The cellobiose dehydrogenase from Neurospora crassa is subjected to codon optimization, so that the cellobiose dehydrogenase is successfully expressed in Pichia pastoris, purified protein is successfully obtained, deglycosylated cellobiose dehydrogenase is successfully obtained after the cellobiose dehydrogenase is treated by endoglycosidase Hf and mannosidase, and a lactose biosensor element capable of directly transferring electrons is successfully prepared for detecting lactose, so that the rapid development of the lactose biosensor is effectively promoted, and the lactose biosensor has a good practical application value.
Description
Technical Field
The invention relates to the technical field of biological enzyme genetic engineering and biosensing, in particular to a deglycosylated cellobiose dehydrogenase, a lactose biosensor and application thereof.
Background
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Lactose is a disaccharide, is present in dairy products such as milk, and lactose-containing foods are considered to be a major source of calcium and are very important in human nutrition. Lactase is a hydrolase responsible for the breakdown of lactose into galactose and glucose monomers. This enzyme is present in the digestive system of humans and its content begins to decline in most children 2-5 years of age. Most adults retain lactase activity at the infant level of only 10%, and therefore some people develop symptoms of lactose dyspepsia after drinking milk and dairy products, which is medically known as lactose intolerance. Lactose intolerance or lactase non-persistence (LNP) affects about 65% of the world population, the frequency of primary lactose intolerance varies greatly between different ethnic and ethnic groups, approximately 5% of northern euros and more than 90% of southeast asians are affected, so that the determination of lactose content is of utmost importance to import and export dairy products in our country. At present, according to the national standard of food safety, the determination of lactose content mainly adopts high performance liquid chromatography, spectrophotometry and ion chromatography. However, these detection methods have various problems such as high analysis cost and complicated operation. The enzyme electrode biosensor method has the characteristics of strong specificity, high sensitivity, good reproducibility and simple operation, and is widely favored.
The enzyme is the most main detection sensitive element in the lactose biosensor, and currently, the lactose biosensor mostly adopts a complex enzyme combined by galactosidase and glucose oxidase and O 2 Based on O as electron transfer mediator 2 Decrease of (2) or H 2 O 2 The amount of production of (2) is detected, but the oxygen content is not well controlled in the method; high redox potential and high background signal are required. Therefore, new electron transfer media replacing O have been sought 2 Or H 2 O 2 However, chemical synthesis of electron mediators involves problems of high price, high toxicity, high difficulty in operation and the like, so that an enzyme is urgently needed to realize Direct Electron Transfer (DET) between electrodes, thereby constructing an ideal biosensor.
Cellobiose Dehydrogenase (CDH) is a typical monomeric protein consisting of a large dehydrogenase Domain (DH) CDH ) And a smaller cytochrome domain (CYT) CDH ) The former contains a non-covalently bound FAD molecule as a cofactor, and the latter contains heme b as a cofactor, which are linked by a flexible linker peptide. CDH catalyzed reaction starts with DH CDH A domain that oxidizes carbohydrates such as cellobiose, cellooligosaccharide, and lactose to the corresponding lactones. Reported to be less than between the enzyme and the electrode surface only
The DET occurs at a relatively fast rate, while the active center of CDH is exposed on the surface of the enzyme molecule, so that it is expected to realize direct electron transfer between the enzyme molecule and the electrode, thereby realizing a simple electrode device avoiding expensive or harmful media. Previous studies have shown that CDH glycosylation is about 9% -16%, and high glycosylation CDH hinders the electron transport distance from the interior to the electrode surface, slowing the DET rate.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a deglycosylated cellobiose dehydrogenase, a lactose biosensor and application thereof. The cellobiose dehydrogenase from Neurospora crassa is subjected to codon optimization, so that the cellobiose dehydrogenase is successfully expressed in Pichia pastoris, purified protein is successfully obtained, deglycosylated cellobiose dehydrogenase is successfully obtained after the cellobiose dehydrogenase is treated by endoglycosidase Hf and mannosidase, and the lactose biosensor element capable of directly transferring electrons is successfully prepared for detecting lactose. The present invention has been completed based on the above results.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided a cellobiose dehydrogenase having a nucleotide sequence encoding the cellobiose dehydrogenase selected from the group consisting of:
(a) A nucleotide sequence shown as SEQ ID NO. 2; or the like, or a combination thereof,
(b) Has at least 80% (including but not limited to 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%) of identity with the nucleotide sequence shown in SEQ ID No.2, and is capable of expressing the cellobiose dehydrogenase in fungi such as Pichia pastoris.
Further, the cellobiose dehydrogenase is deglycosylated cellobiose dehydrogenase, and specifically, the deglycosylated cellobiose dehydrogenase is obtained by treating the cellobiose dehydrogenase with alpha-1, 2-mannosidase and endoglycosidase Hf. The CDH of the original cellobiose dehydrogenase is about 110KDa, the CDH after deglycosylation is about 90KDa, and the glycosylation degree is about 18 percent.
In a second aspect of the present invention, there is provided a recombinant expression vector containing a nucleotide encoding the cellobiose dehydrogenase as described above.
In a third aspect of the present invention, there is provided a host cell comprising the recombinant vector of the second aspect of the present invention or capable of expressing the cellobiose dehydrogenase as described above.
The host cell may be a prokaryotic cell or a eukaryotic cell; preferably eukaryotic cells, including fungal cells.
The fungal cell comprises a yeast. Particularly, pichia pastoris has the advantages of clear genetic background, high foreign protein expression level, capability of correctly folding, modifying and processing protein, capability of performing high-density fermentation and the like, and can be used as an excellent expression host of foreign protein, so the pichia pastoris is preferably used as a host cell.
In a fourth aspect of the present invention, there is provided an application of the cellobiose dehydrogenase, the recombinant expression vector or the host cell described above in lactose detection or lactose biosensor preparation.
In a fifth aspect of the present invention, there is provided a lactose biosensor comprising the cellobiose dehydrogenase.
Specifically, the lactose biosensor contains an enzyme electrode comprising:
a base electrode, and a base material carried by the base electrode.
The substrate electrode includes, but is not limited to: a Glassy Carbon (GCE) electrode, a gold electrode, a graphite electrode, a carbon paste electrode, preferably GCE. The GCE has good mechanical stability, light stability and high conductivity.
The substrate material includes the cellobiose dehydrogenase and the carbon material.
In a sixth aspect of the invention, there is provided a method of electrochemically measuring the concentration or presence of lactose, the method comprising: the sensor is contacted with a liquid sample having or suspected of having lactose, the response current intensity of the target analyte to be measured is measured, and the concentration or presence of lactose is analyzed.
The beneficial technical effects of the technical scheme are as follows:
the technical scheme comprises the steps of carrying out codon optimization on cellobiose dehydrogenase from Neurospora crassa, so that the cellobiose dehydrogenase is successfully expressed in Pichia pastoris, successfully obtaining purified protein, treating with endoglycosidase and mannosidase, obtaining the deglycosylated cellobiose dehydrogenase, and successfully preparing a lactose biosensor element capable of directly carrying out electron transfer for detecting lactose, so that the rapid development of the lactose biosensor is effectively promoted, and the lactose biosensor has a good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 shows SDS-PAGE detecting different concentrations of imidazole eluting recombinant expression cellobiose dehydrogenase in the present example; in the figure, 5mM-100mM represent the concentration of imidazole, and lanes 1-6 represent cellobiose dehydrogenase eluents obtained in the order of 1 collection tube per 1mL of tube when eluting recombinant proteins with a specific concentration of imidazole.
FIG. 2 is a comparison graph of the electrophoresis of purified raw cellobiose dehydrogenase and deglycosylated cellobiose dehydrogenase in this example; in the figure, 1 is Nc _ CDH having a molecular weight of about 110kDa, and 2 is 600 mL-concentrated Nc _ dCDH having a molecular weight of about 90 kDa.
FIG. 3 is a graph showing a comparison of optimum temperature and optimum pH between Nc _ CDH and Nc _ dCDH in examples of the present invention; a graph showing the results of enzyme activity changes under different reaction temperature conditions of Nc _ CDH and Nc _ dCDH; graph showing the results of enzyme activity changes under different pH reaction conditions of Nc _ CDH and Nc _ dCDH.
FIG. 4 shows multi-walled carbon nanotubes of different ratios in an embodiment of the present invention: a cyclic voltammogram of the Nc _ CDH modified glassy carbon electrode as a function of lactose concentration; a.0.1% carbon tube 5. Mu.L, 5. Mu.L enzyme, 7. Mu.L; b, 10 μ L of 0.1% carbon tube, 10 μ L of enzyme, 14 μ L; c.5% carbon tube 5. Mu.L enzyme, 7. Mu.L; d.0.5% carbon tube 10. Mu.L, 10. Mu.L enzyme, 14. Mu.L; e.1% carbon tube 5. Mu.L enzyme, 7. Mu.L; f.1% carbon tube 10. Mu.L, 10. Mu.L enzyme, 14. Mu.L; g.5% carbon tube 5. Mu.L enzyme, 7. Mu.L; h.1.5% carbon tube 10. Mu.L enzyme, 14. Mu.L.
FIG. 5 is a Cyclic Voltammetry (CV) graph, square Wave Voltammetry (SWV) graph and correlation analysis for Nc _ CDH and Nc _ dCDH modified electrodes at a gradient lactose concentration in an example of the present invention. Nc _ CDH modified electrode CV diagram at 5-40mmol/L lactose concentration gradient; b. CV plot of deglycosylated Nc _ dCDH at 5-40mmol/L lactose concentration gradient; SWV plot of Nc _ CDH modified electrode at 5-80mmol/L lactose concentration gradient; SWV plots of Nc _ dCDH modified electrodes at 5-80mmol/L lactose concentration gradient; graph analyzing linear correlation of peak potential value of square wave voltammetry curve and sugar concentration under 5-80mmol/L lactose concentration gradient by Nc _ CDH modified electrode; f. graph analyzing the linear correlation of peak potential value of square wave voltammogram and sugar concentration under 5-80mmol/L lactose concentration gradient of deglycosylated Nc _ dCDH.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
Currently, the most important CDH production strains in the industry are Venturia crassa and Venturia sanguinea, but the CDH production yield is low, and the extraction and purification process is complicated, so that the construction of more excellent and high-yield CDH production strains by using a genetic engineering means is always the direction of research efforts of researchers. The pichia pastoris has the advantages of clear genetic background, high foreign protein expression level, capability of correctly folding, modifying and processing the protein, capability of performing high-density fermentation and the like, thereby being used as an excellent expression host of the foreign protein.
In view of the above, in one exemplary embodiment of the present invention, there is provided a cellobiose dehydrogenase whose nucleotide sequence encoding the cellobiose dehydrogenase is selected from the group consisting of:
(a) A nucleotide sequence shown as SEQ ID NO. 2; or the like, or a combination thereof,
(b) Has at least 80% (including but not limited to 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%) or more identity to the nucleotide sequence represented by SEQ ID NO.2, and is capable of expressing the cellobiose dehydrogenase in fungi such as Pichia pastoris.
In another embodiment of the present invention, the cellobiose dehydrogenase is a deglycosylated cellobiose dehydrogenase, and specifically, the deglycosylated cellobiose dehydrogenase is obtained by treating the cellobiose dehydrogenase with α -1, 2-mannosidase and endoglycosidase Hf. The CDH of the original cellobiose dehydrogenase is about 110KDa, the CDH after deglycosylation is about 90KDa, and the glycosylation degree is about 18 percent.
In another embodiment of the present invention, there is provided a recombinant expression vector comprising a nucleotide encoding the cellobiose dehydrogenase as described above.
Specifically, the recombinant expression vector is obtained by operatively linking the nucleotide molecule to an expression vector, wherein the expression vector is any one or more of a viral vector, a plasmid, a phage, a cosmid or an artificial chromosome; in one embodiment of the invention, the expression vector is a plasmid; further, the plasmid is a pPIC9K vector.
In still another embodiment of the present invention, there is provided a host cell comprising the recombinant vector according to the second aspect of the present invention or capable of expressing the cellobiose dehydrogenase as described above.
The host cell may be a prokaryotic cell or a eukaryotic cell; preferably eukaryotic cells, including fungal cells.
Wherein the bacterial cell is any of the genera Escherichia, agrobacterium, bacillus, streptomyces, pseudomonas, or Staphylococcus;
more specifically, the bacterial cell is Escherichia coli (e.g., escherichia coli DH 5. Alpha.), agrobacterium tumefaciens, agrobacterium rhizogenes, lactococcus lactis, bacillus subtilis, bacillus cereus, or Pseudomonas fluorescens.
The fungal cell comprises a yeast. Particularly, pichia pastoris has the advantages of clear genetic background, high foreign protein expression level, capability of correctly folding, modifying and processing protein, capability of performing high-density fermentation and the like, and can be used as an excellent expression host of foreign protein, so the pichia pastoris is preferably used as a host cell in the invention.
In another embodiment of the present invention, there is provided a use of the cellobiose dehydrogenase, the recombinant expression vector or the host cell described above in lactose detection or in the preparation of a lactose biosensor.
In another embodiment of the present invention, there is provided a lactose biosensor comprising the cellobiose dehydrogenase.
Specifically, the lactose biosensor contains an enzyme electrode comprising:
a base electrode, and a base material carried by the base electrode.
The substrate electrode includes, but is not limited to: a Glassy Carbon (GCE) electrode, a gold electrode, a graphite electrode, a carbon paste electrode, preferably GCE. The GCE has good mechanical stability, light stability and high conductivity.
The substrate material comprises the cellobiose dehydrogenase and a carbon material, and the carbon material comprises but is not limited to: the electrode material comprises activated carbon, graphene, carbon nanofibers, carbon nanospheres, glassy carbon, carbon aerogel and Carbon Nanotubes (CNTs), and preferably the carbon nanotubes have large specific surface area and small internal resistance, so that the analysis performance of the chemically modified electrode can be remarkably improved, and the electrode material has good conductivity and chemical stability.
The carbon nano tube not only comprises a multi-wall carbon nano tube and a single-wall carbon nano tube, but also comprises a functionalized carbon nano tube modified by amination, carboxylation and the like.
In a specific embodiment of the present invention, the enzyme electrode comprises: a GCE electrode, and cellobiose dehydrogenase and multi-wall carbon nanotubes loaded on the GCE electrode. The loading may be performed by means of dispensing, and the like, and is not particularly limited herein.
More specifically, the lactose biosensor comprises at least two electrodes, which comprise at least the above-mentioned enzyme electrode.
In another embodiment of the present invention, in the lactose biosensor, the lactose biosensor comprises two or three electrodes, and accordingly, the lactose biosensor is a two-electrode or three-electrode sensor.
In yet another embodiment of the present invention, in a sensor consisting of two electrodes (i.e., a two-electrode sensor), the electrodes are a working electrode and a counter electrode; wherein the working electrode is the enzyme electrode.
In yet another embodiment of the present invention, in a sensor consisting of three electrodes (i.e., a three-electrode sensor), the electrodes are a working electrode, a counter electrode, and a reference electrode; wherein the working electrode is the enzyme electrode.
In yet another embodiment of the present invention, in the three-electrode enzyme sensor, the counter electrode may be a platinum electrode; the reference electrode may be an Ag/AgCl electrode, and is not particularly limited herein.
In yet another embodiment of the present invention, there is provided a method of electrochemically measuring the concentration or presence of lactose, the method comprising: the sensor is contacted with a liquid sample having or suspected of having lactose, the response current intensity of the target analyte to be measured is measured, and the concentration or presence of lactose is analyzed.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
1. Cellobiose dehydrogenase gene codon optimization and recombinant expression plasmid construction
Cellobiose dehydrogenase gene XM-951498 (CDH II A) in Neurospora crassa OR74A genome is used as an original gene, the gene sequence is shown as SEQ ID NO.1, the cellobiose dehydrogenase gene is optimized according to the codon preference of a pichia pastoris expression host, and the optimized sequence is shown as SEQ ID NO. 2. And synthesizing the optimized cellobiose dehydrogenase gene, and connecting the cellobiose dehydrogenase gene to the middle of EcoRI and NotI enzyme cutting sites in a pPIC9K vector based on the principle of homologous recombination to obtain a cellobiose dehydrogenase recombinant expression plasmid.
2. Cellobiose dehydrogenase recombinant expression plasmid transformed Escherichia coli (DH 5 alpha) enriched plasmid
Mixing the cellobiose dehydrogenase recombinant expression plasmid with E.coli DH5 alpha competent cells, standing on ice for 30min, thermally shocking for 45s, standing on ice for 2min, coating the mixture on an LB agar culture plate with 100 mu g/mL ampicillin resistance, culturing overnight at 37 ℃, extracting the plasmid by using a plasmid extraction kit, and realizing the enrichment of the cellobiose dehydrogenase recombinant expression plasmid.
3. Transformation of recombinant expression plasmid of cellobiose dehydrogenase into host bacterium of pichia pastoris
Pichia pastoris GS115 was inoculated into a single colony in a test tube containing 5mL of YPD liquid medium and cultured overnight at 30 ℃. Inoculating into a triangular flask containing 50mL YPD liquid culture medium according to the inoculation amount of 1%, and culturing overnight at 30 ℃ until OD600= 1.3-1.5; the culture solution was centrifuged at 1500g and 4 ℃ for 5min, the supernatant was discarded, and the cells were resuspended in 50mL ice-bath double distilled water; 1500g, centrifuging the culture solution at 4 deg.C for 5min, discarding the supernatant, and resuspending the cells with 25mL ice-bath double distilled water; 1500g, centrifuging the culture solution at 4 deg.C for 5min, discarding the supernatant, and resuspending the cells with 2mL of ice-cooled 1M sorbitol solution; the culture solution was centrifuged at 1500g,4 ℃ for 5min, the supernatant was discarded, and the cells were resuspended in 1mL of ice-cooled 1M sorbitol solution to a suspension volume of approximately 1.5mL; adding 80 mu L of the treated competent cells and 5-20 mu g of recombinant expression plasmid of cellobiose dehydrogenase obtained by SacI linearization into a 1.5mL precooled centrifuge tube, and uniformly mixing. Then the mixed solution is transferred into a transformation cup (0.2 cm type) which is pre-iced; ice-bath the transformation cup containing transformation mixture for 5min; setting an electrotransformation instrument (Voltage (V): 2000; capacitance (uF): 25; resistance (omega): 200; cuvette (mm): 2) according to the electrotransfer parameters of the Biorad Pichia pastoris, starting electric pulse, immediately adding 1mL of ice-bath 1M sorbitol solution into a transformation cup after the pulse, and then transferring the transformation solution into 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.
4. Cellobiose dehydrogenase gene induced expression
Inoculating the screened pichia pastoris recombinant into 30mL YPD liquid culture medium, performing shaking culture at 30 ℃ and 250rpm for overnight; transfer 50 μ L of overnight culture into 50mL BMGY liquid medium at 30 ℃,250rpm, shake culture to OD600= 2-6 (logarithmic growth phase, approximately 16-18 h); 3000g, centrifuging for 5min, discarding the supernatant, resuspending the cells with BMMY liquid medium to OD600=1.0, continuously adding methanol (final concentration of methanol is 1%) for 5 days, placing in a 500mL triangular flask, sealing with 8 layers of sterilized gauze, performing shaking culture at 30 ℃,250 rpm; SDS-PAGE detects whether the foreign gene is expressed or not; the results showed that cellobiose dehydrogenase gene (XM-951498) was successfully expressed in Pichia pastoris.
5. Deglycosylation and separation purification of cellobiose dehydrogenase
Alpha-1, 2-mannosidase and endoglycosidase Hf (New England Biolabs, ipswich, MA, U.S.A.) were used as per fermentation broths: hf: the purified glycosylated Nc _ CDH was treated with α -1, 2-mannosidase (30.
Mixing the crude enzyme solution with target protein expression detected by SDS-PAGE and filler containing nickel, and rotating and combining for 6h in a refrigerator at 4 ℃. Then, the hetero-protein was eluted with imidazole solutions at concentrations of 5mM and 10mM, the objective cellobiose dehydrogenase was eluted with 20mM-250mM imidazole solution, and the objective protein was detected by SDS-PAGE (FIG. 1). The protein solution eluted with 30mM and 40mM imidazole was ultrafiltered with disodium hydrogenphosphate-citric acid buffer solution of pH 5.0 at 8000rpm,3min,4 ℃ until the pH of the running-down buffer solution was 5.0, the ultrafiltration was stopped, and the enzyme solution obtained by the ultrafiltration was collected. FIG. 1 shows the electrophoretogram of recombinant proteins eluted with different concentrations of imidazole after the successful expression of deglycosylated cellobiose dehydrogenase gene in Pichia pastoris, and it can be seen from FIG. 1 that purified cellobiose dehydrogenase can be obtained by elution with 30mM and 40mM imidazole. FIG. 2 shows the spectra of the original cellobiose dehydrogenase and deglycosylated cellobiose dehydrogenase after concentration and purification. As shown by picture comparison, the original CDH is about 110KDa, the CDH after deglycosylation is about 90KDa, and the glycosylation degree is about 18%.
6. Comparison of original Nc _ CDH with deglycosylated Nc _ dCDH enzymology
The enzyme activity was measured by adding Nc _ CDH and Nc _ dCDH at equal concentrations to buffer systems of different pH values, each of which was a citric acid-phosphoric acid buffer (pH 3.0, 4.0, 4.5, 5.0, 6.0, 7.0 and 8.0) at a concentration of 100mmol/L, at an enzyme reaction temperature of 30 ℃ for 3min. As a result of determining the optimum pH, as shown in FIG. 3, both Nc _ CDH and Nc _ dCDH maintained high activity at pH 4-7, and the enzyme activity decreased sharply at pH lower than 4 or higher than 7.
The enzyme activity of Nc _ CDH and Nc _ dCDH at the optimum reaction temperature was measured under the condition of 100mmol/L citric acid-phosphate buffer (optimum pH) for 3min at a temperature of 20 ℃ to 60 ℃ at every 10 ℃. As a result of the measurement of the optimum temperature, as shown in FIG. 3, the optimum reaction temperatures of Nc _ CDH and Nc _ dCDH were 30 ℃ and 40 ℃, respectively, and the temperature adaptability of the enzyme molecule was improved after deglycosylation.
7. Preparation of multi-wall carbon nano-tube-enzyme modified glassy carbon electrode
Pretreating a bare glassy carbon electrode: polishing the glassy carbon electrode by firstly polishing the glassy carbon electrode
Washing with deionized water, and sequentially using
And 50 μm Al 2 O 3 Polishing the polishing powder until the surface of the electrode is polished, rinsing the electrode with distilled water, ultrasonically cleaning the electrode for 1min, taking out the electrode, naturally drying the electrode, ultrasonically cleaning the electrode with nitric acid (V: V = 1).
Cyclic voltammetric scanning in potassium ferricyanide solution: weighing 0.0823g potassium ferricyanide and 5.055g KNO 3 Placing in 250mL beaker, adding distilled water, stirring to dissolve, transferring to 250mL volumetric flask, shaking to constant volume to obtain 1.0 × 10 -3 mol/L potassium ferricyanide solution (containing 0.2mol/L KNO) 3 ) And scanning the glassy carbon electrode in the prepared potassium ferricyanide solution by using cyclic voltammetry, wherein the potential difference is within 64mV-80 mV.
GCE electrode (diameter: 3 mm) was used as working electrode, multi-walled carbon nanotubes (S-MWNT, 1 mg) were added to 0.1% aqueous Nafion solution (1 mL) and sonicated for 2h until a black homogeneous suspension (1 mg/mL) was successfully obtained. S-MWNT/Nc _ CDH modified GCE was obtained by transferring 14. Mu.L of a suspension of the enzyme mixed with carbon nanotubes to the surface of freshly polished GCE and allowing the electrodes to air dry. After natural drying, 2. Mu.L of 0.5% Nafion solution was added dropwise to the surface, and after natural drying, the mixture was left in a refrigerator at 4 ℃ overnight for further use. S-MWNT/Nc _ dCDH was obtained in the same manner.
8. Cellobiose dehydrogenase electrode electrochemical characterization condition optimization
The electrochemical properties of the S-MWNT/Nc-CDH modified electrode are detected and researched by adopting a three-electrode system, the potential range is-0.6-0.5V, and the same enzyme modified electrode is scanned from 0.01V/S to 1.0V/S respectively for researching the influence of the scanning rate on electrode signals. Along with the increase of the scanning speed, the current signal is more and more obvious, and when the scanning speed exceeds 0.1V/s, the fluctuation of the current signal is larger, so that the subsequent experiment selects 0.1V/s as the electrode scanning speed.
Optimizing the concentration of the carbon tubes and the enzyme amount ratio under the conditions that a is 0.1 percent, 5 mu L of the carbon tubes is 5 mu L, and 7 mu L of the enzyme is taken; b, taking 14 mu L of 0.1 percent carbon tube 10 mu L and 10 mu L enzyme; c, 0.5 percent of carbon tube, 5 mu L of enzyme, and taking 7 mu L of enzyme; d, 0.5 percent of carbon tube 10 mu L,10 mu L of enzyme, taking 14 mu L; e, taking 7 mu L of 1% carbon tube 5 mu L and 5 mu L enzyme; f, taking 14 mu L of 1% carbon tube 10 mu L and 10 mu L enzyme; g: 5 mul of 1.5% carbon tube 5 mul of enzyme, 7 mul; h 10. Mu.L of 1.5% carbon tube and 10. Mu.L enzyme, 14. Mu.L was taken. As shown in FIG. 4, the results were the best results, the highest current response and the most significant redox peak, using 1% carbon tubes and 10. Mu.L of 10. Mu.L enzyme.
9. Measurement of electrochemical parameters of Cellobiose dehydrogenase electrode
The electrochemical properties of the enzyme electrode are detected and researched by adopting a three-electrode system, and a bare glassy carbon electrode, an S-MWNT/Nc _ CDH modified electrode and an S-MWNT/Nc _ dCDH modified electrode are subjected to Cyclic Voltammetry (CV) scanning on lactose solutions with different concentrations (5 mmol/L,10mmol/L,15mmol/L,20mmol/L,25mmol/L,30mmol/L,35mmol/L and 40 mmol/L) by using cyclic voltammetry in sequence at a potential range of-0.6-0.5V, and the results show that the two enzyme electrodes show the oxidation reaction characteristics that the oxidation peak is gradually increased and the reduction peak is gradually reduced along with the increase of the lactose concentration, but the current value and the linear gradient response of the Nc _ dCDH modified electrode are obviously higher than those of the Nc _ CDH modified electrode (FIGS. 5a and 5b). In order to further obtain more accurate sensitivity and detection limit and be better applied to a biosensor, two enzyme electrodes are researched by using square wave voltammetry SWV, and the result shows that under the condition of a concentration gradient of 5 mM-80 mM, the linear equation of Nafion/S-MWNT/Nc _ CDH/GCE is y =2.644e -8 *x+1.776e -7 ，R 2 =0.9990, and it is calculated that when the electrode signal to noise ratio is 3 (S/N = 3), the limit of detection is 4.015mM, the sensitivity is 37.424 μ a.mmol L -1 .cm -2 Under the same substrate concentration gradient, the Nafion/S-MWNT/Nc _ dCDH/GCE electrode linear regression equation is y =2.782e -8 *x+3.715e -7 *x，R 2 =0.9995, its sensitivity 39.377 μ A.mmol L -1 .cm -2 The detection limit is 2.006mM (S/N = 3), and the deglycosylated Neurospora crassa CDH enzyme electrode sensitivity is improved compared with the original enzyme, and the detection limit is higher than the original enzymeThe starting enzyme electrode is about one time lower, which shows that the electron transfer efficiency between the enzyme and the electrode is increased after the cellobiose dehydrogenase is deglycated, and the construction of the third-generation lactose biosensor is more facilitated.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A cellobiose dehydrogenase, wherein the nucleotide sequence encoding said cellobiose dehydrogenase is selected from the group consisting of:
(a) A nucleotide sequence shown as SEQ ID NO. 2; or the like, or, alternatively,
(b) Has at least more than 80 percent of identity with the nucleotide sequence shown in SEQ ID NO.2, and can express the cellobiose dehydrogenase in pichia pastoris.
2. The cellobiose dehydrogenase according to claim 1, wherein the cellobiose dehydrogenase is a deglycosylated cellobiose dehydrogenase obtained by treating the cellobiose dehydrogenase with α -1, 2-mannosidase and endoglycosidase Hf.
3. A recombinant expression vector comprising a nucleotide encoding the cellobiose dehydrogenase of claim 1.
4. The recombinant expression vector of claim 3, wherein the recombinant expression vector is obtained by operably linking the nucleotide to an expression vector, which is any one or more of a viral vector, a plasmid, a phage, a cosmid, or an artificial chromosome; further, the plasmid is a pPIC9K vector.
5. A host cell comprising the recombinant vector of claim 2 or capable of expressing the cellobiose dehydrogenase of claim 1 or 2.
6. The host cell of claim 5, wherein the host cell is a prokaryotic cell or a eukaryotic cell; the eukaryotic cell is pichia pastoris.
7. Use of the cellobiose dehydrogenase of claim 1 or 2, the recombinant expression vector of claim 3 or 4, or the host cell of claim 5 or 6 for lactose detection or for the preparation of a lactose biosensor.
8. A lactose biosensor, which contains cellobiose dehydrogenase as claimed in claim 1 or 2.
9. The lactose biosensor of claim 8 which contains an enzyme electrode comprising:
a base electrode, and a base material carried by the base electrode;
the base electrode includes: glassy carbon electrodes, gold electrodes, graphite electrodes, carbon paste electrodes;
the substrate material comprising the cellobiose dehydrogenase of claim 1 or 2 and a carbon material comprising: activated carbon, graphene, carbon nanofibers, carbon nanospheres, glassy carbon, carbon aerogel, carbon nanotubes;
further, the enzyme electrode includes: a glassy carbon electrode, cellobiose dehydrogenase and a multi-walled carbon nanotube which are loaded on the glassy carbon electrode;
further, the lactose biosensor is a two-electrode or three-electrode sensor; wherein the working electrode is the enzyme electrode.
10. A method of electrochemically measuring the concentration or presence of lactose, the method comprising: contacting the lactose biosensor of claim 8 or 9 with a liquid sample having or suspected of having lactose, measuring the intensity of the response current of lactose, and analyzing the concentration or presence of lactose.
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