Glucose dehydrogenase mutant pTrc99A-6, preparation method and application thereof, and blood glucose test paper

Abstract

The invention provides a glucose dehydrogenase mutant pTrc99A-6 as well as a preparation method and application thereof and blood glucose test paper, and relates to the technical field of biology. The amino acid sequence of the glucose dehydrogenase mutant pTrc99A-6 provided by the invention is as shown in SEQ ID NO. 1. Compared with a wild type, the glucose dehydrogenase mutant has better stability and more specific substrate specificity, and can be used for preparing a blood glucose detection product.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a glucose dehydrogenase mutant pTrc99A-6 and a preparation method, application and blood sugar detection test strip thereof. Background Art

[0002] Glucose dehydrogenase (GDH) is an oxidoreductase that can catalyze the dehydrogenation reaction of glucose to generate gluconolactone. During the reaction, glucose loses electrons and transfers them to the coenzyme or prosthetic group of GDH. The content of the reduced prosthetic group or coenzyme generated is positively correlated with the concentration of glucose, so it can be used for blood sugar detection, and the detection is not interfered by the general concentration of anticoagulants, preservatives, uric acid, bilirubin, etc. Compared with the traditional GOD method and HK method, the GDH method is simpler to operate, and only one enzyme is required to participate in the reaction. It can also be used for continuous monitoring (λ=340nm) and endpoint analysis, so it is often used for blood sugar determination.

[0003] GDH can be divided into three types according to the different cofactors or coenzymes it binds: PQQ-dependent GDH (PQQ-GDH), NAD-dependent GDH (NAD-GDH), and FAD-dependent GDH (FAD-GDH). The soluble expressed sPQQ-GDH has poor substrate specificity and a wide substrate spectrum; the membrane-bound mPQQ-GDH is a membrane protein, which is difficult to express and separate and purify, all of which severely limit its application in blood glucose determination. NAD-GDH needs to continuously add cofactors to maintain its activity due to its weak cofactor binding, which is not conducive to blood glucose detection. The cofactor of FAD-GDH is tightly bound and not easy to fall off. Secondly, oxygen is not required during the reaction process. Therefore, both the accuracy of detection and the long-term storage are better than glucose oxidase and other cofactor-dependent GDH. In addition, the core enzyme used by the mainstream blood glucose meters on the market is also FAD-dependent glucose dehydrogenase, which can generally be used from the time of opening until the end of the validity period.

[0004] Wild-type FAD-GDH from different sources generally has multiple substrate activities and still has certain dehydrogenation and oxidation activity towards maltose and xylose, but has poor specificity. If such components exist in the blood, false blood sugar will appear when blood sugar is tested.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The first object of the present invention is to provide a glucose dehydrogenase (FAD-GDH) mutant pTrc99A-6, which has good stability and substrate specificity, so as to solve the above technical problems.

[0007] The second object of the present invention is to provide a nucleotide.

[0008] The third object of the present invention is to provide a carrier.

[0009] A fourth object of the present invention is to provide a cell.

[0010] The fifth object of the present invention is to provide a method for preparing the glucose dehydrogenase mutant pTrc99A-6.

[0011] The sixth object of the present invention is to provide the use of the above-mentioned glucose dehydrogenase mutant pTrc99A-6 in the preparation of blood glucose detection products.

[0012] A seventh object of the present invention is to provide a blood sugar test strip.

[0013] In order to achieve the above objectives, the following technical solutions are proposed:

[0014] In a first aspect, the present invention provides a glucose dehydrogenase mutant pTrc99A-6, the amino acid sequence of the glucose dehydrogenase mutant pTrc99A-6 is shown in SEQ ID NO.1.

[0015] In a second aspect, the present invention provides a nucleotide comprising a nucleotide sequence encoding the above-mentioned glucose dehydrogenase mutant pTrc99A-6.

[0016] As a further technical solution, the sequence of the nucleotide is shown as SEQ ID NO.2.

[0017] In a third aspect, the present invention provides a vector carrying the nucleotide.

[0018] As a further technical solution, the vector includes a plasmid.

[0019] In a fourth aspect, the present invention provides a cell, wherein the cell carries the nucleotide, or contains the vector, or expresses the glucose dehydrogenase mutant pTrc99A-6.

[0020] As a further technical solution, the cells include Escherichia coli and Pichia pastoris.

[0021] In a fifth aspect, the present invention provides a method for preparing a glucose dehydrogenase mutant pTrc99A-6, which is obtained by fermentation of the above-mentioned cells.

[0022] In a sixth aspect, the present invention provides the use of the above-mentioned glucose dehydrogenase mutant pTrc99A-6 in the preparation of blood glucose detection products.

[0023] In a seventh aspect, the present invention provides a blood glucose test strip, which uses a glucose dehydrogenase method to detect blood glucose; the glucose dehydrogenase in the blood glucose test strip is the glucose dehydrogenase mutant pTrc99A-6.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The glucose dehydrogenase mutant pTrc99A-6 provided by the present invention has better stability and more specific substrate specificity than the wild type, and can be used to prepare blood sugar detection products. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 The color development results of different monoclonal bacterial solutions on the ELISA plate (10 minutes). DETAILED DESCRIPTION

[0028] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] The term "vector" refers to a nucleic acid delivery vehicle into which a nucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell.

[0030] In a first aspect, the present invention provides a glucose dehydrogenase mutant pTrc99A-6, the amino acid sequence of the glucose dehydrogenase mutant pTrc99A-6 is shown in SEQ ID NO.1:

[0031] (SEQ ID NO.1).

[0032] The inventors mutated the amino acids at positions 336 and 555 of wild-type glucose dehydrogenase and obtained a glucose dehydrogenase mutant pTrc99A-6. The inventors found that the mutant had better stability and more specific substrate specificity than the wild-type.

[0033] In a second aspect, the present invention provides a nucleotide comprising a nucleotide sequence encoding the above-mentioned glucose dehydrogenase mutant pTrc99A-6.

[0034] The nucleotide can be expressed and translated to obtain the glucose dehydrogenase mutant pTrc99A-6 of the present invention.

[0035] In some optional embodiments, the nucleotide sequence is shown in SEQ ID NO.2:

[0036]

[0037] In a third aspect, the present invention provides a vector carrying the nucleotide.

[0038] By introducing the vector into the recipient cells, the recipient cells can express the glucose dehydrogenase mutant pTrc99A-6 of the present invention.

[0039] In some optional embodiments, the vector includes but is not limited to a plasmid.

[0040] In a fourth aspect, the present invention provides a cell, wherein the cell carries the nucleotide, or contains the vector, or expresses the glucose dehydrogenase mutant pTrc99A-6.

[0041] In some optional embodiments, the cells include but are not limited to Escherichia coli and Pichia pastoris, and may also be other cells well known to those skilled in the art.

[0042] In a fifth aspect, the present invention provides a method for preparing a glucose dehydrogenase mutant pTrc99A-6, which is obtained by fermentation of the above-mentioned cells.

[0043] The preparation method is simple and efficient, and a large amount of glucose dehydrogenase mutant pTrc99A-6 can be obtained through fermentation.

[0044] In a sixth aspect, the present invention provides the use of the above-mentioned glucose dehydrogenase mutant pTrc99A-6 in the preparation of blood glucose detection products.

[0045] The glucose dehydrogenase mutant pTrc99A-6 provided by the present invention has better stability and more specific substrate specificity than the wild type, and can be used to prepare blood sugar detection products.

[0046] In a seventh aspect, the present invention provides a blood glucose test strip, which uses a glucose dehydrogenase method to detect blood glucose; the glucose dehydrogenase in the blood glucose test strip is the glucose dehydrogenase mutant pTrc99A-6.

[0047] Since the glucose dehydrogenase mutant pTrc99A-6 of the present invention has better specificity, the blood sugar test strip prepared by using the mutant has higher accuracy.

[0048] The present invention is further described below by means of specific examples and comparative examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0049] Example 1

[0050] 1. Determination of glucose dehydrogenase mutants:

[0051] The name of the plasmid with the original FAD-GDH sequence selected in this study is pTrc99a-WT (NcoI / Xhol), and the amino acid sequence of FAD-GDH is as follows:

[0052] TSAKYDYIVIGGGTSGLAVANRLSEDPSVNVLILEAGGSVWNNPNV

[0053] TNVNGYGLAFGSDIDWQYQSVNQPYGGNVSQVLRAGKALGGTSTING

[0054] MAYTRAEDVQIDAWETIGNTGWTWKNLFPYYRKSENFTVPTKSQTSLG

[0055] ASYEAGAHGHEGPLDVAFTQIESNNLTTYLNRTFQGMGLPWTEDVNGG

[0056] KMRGFNLYPSTVNLEEYVREDAARAYYWPYKSRPNLHVLLNTFANRIV

[0057] WDGEARDGDITASGVEITSRNGTVRVINAEKEVIVSAGALKSPAILELSGI

[0058] GNPSVLDKYNIPVKVNLPTVGENLQDQVNSHMDASGNTSISGTKAVSYP

[0059] DVYDVFGDEAESVAKQIRANLKQYAADTAKANGNIMKAADLERLFEVQ

[0060] YDLIFKGRVPIAEVLNYPGSATSVFAEFWALLPFARGSVHIGSSNPAEFPVI

[0061] NPNYFMLDWDAKSYVAVAKYIRRSFESYPLSSIVKESTPGYDVIPRNASE

[0062] QSWKEWVFDKNYRSNFHPVGTAAMMPREIGGVVDERLNVYGTTNVRV

[0063] VDASVLPFQVCGHLVSTLYAVAERAADLIKADAGRR(SEQ ID NO.3).

[0064] In this study, we first compared the GOD crystal structure 1CF3 downloaded from the PDB database, and used the software PyMOL to superimpose the structure with the wild-type FAD-GDH. We searched for the active center residues of FAD-GDH based on the conserved residues in the active center of GOD, and compared the differences in the structures around the active sites of the two structures. Based on the structural comparison of FAD-GDH and GOD, we used the Autodock software for molecular docking to obtain the substrate binding pocket model of FAD-GDH and D-glucose, and analyzed the interaction between the enzyme and the substrate. The mutation site was determined based on the model and the results of the previous structural comparison.

[0065] The selected mutation points are shown in Table 1, and corresponding mutation primers were designed according to each mutation site.

[0066] Table 1 Selected mutation sites of FAD-GDH and corresponding mutation primers

[0067]

[0068] The above 6 primers were synthesized by a primer synthesis company, and the mutation of the above sites was completed using a commercial mutation kit according to the instructions.

[0069] After completing the above mutation, the obtained plasmid was transformed into E. coli DH5α competent cells, spread on LB (containing Amp resistance) plates, and cultured overnight at 37°C. When white colonies grow on the LB plates, the colonies were picked up and expanded in 96 deep-well plates and cultured at 37°C for 4-6 hours.

[0070] The above bacterial solution was inoculated into a new 96-deep-well plate at a density of 5%, and an appropriate amount of autoinduction medium (20 g / L tryptone, 5 g / L yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 0.5 g / L glucose, 2 g / L lactose, 1 x trace element, 10 mM potassium phosphate buffer, pH 7.0) was added. After culturing at 37°C for 4 hours, the culture was then cultured at 20°C for 24 hours. After the culture was completed, the 96-deep-well plate was left to stand at 4°C for 3 hours, and then the supernatant was carefully discarded. The 96-deep-well plate was then sealed and placed in a 50°C oven for 0.5 hours. Transfer 20ul of the bacterial solution at the bottom of the deep-well plate to a new 96-well ELISA plate, add 280ul of reaction solution (100mM potassium phosphate buffer, pH7.0, containing 100mM D-glucose, 0.6mM PMS (phenazine methyl sulfate), 0.12mM DCIP (2,6-dichloroindophenol sodium), 0.1% TritonX-100), incubate on a ELISA reader at 37°C for 5 minutes, then measure the OD600 value every 1 minute, with an interval of 10s between each measurement, and measure 5-10 times in total. Finally, screen the mutant colonies based on the color of the ELISA plate and the difference in OD600. The color development results of the ELISA plate are as follows: Figure 1 shown.

[0071] According to the color change results of the above ELISA plate and the difference in OD600, a monoclonal strain was selected and sent to a sequencing company for sequencing. The sequencing results are shown in Table 2.

[0072] Table 2 Summary of sequencing results of monoclonal strains sent for sequencing

[0073] Mutant name Gene mutation content pTrc99A-52 Y118W pTrc99A-118 K118D pTrc99A-118 K118E pTrc99A-160 T160R pTrc99A-336 Y336F pTrc99A-340 Y340F pTrc99A-555 V555Y pTrc99A-555 V555M

[0074] Use the software BioPro to predict the 1 / 2 / 3 site mutations of the above sites, and select the combination with higher scores to send to the company for synthesis. After obtaining the plasmid, transform the plasmid into the competent cells of Escherichia coli BL21 (DE3), spread it on the LB (containing Amp resistance) plate, and culture it at 37°C overnight. When white colonies grow on the LB plate, select 5 monoclonal bacteria from each plate for expansion culture, and culture them at 37°C for 4-6 hours.

[0075] The above bacterial solution was inoculated into a 96-deep-well plate at a density of 5%, and an appropriate amount of autoinduction medium was added. After culturing at 37°C for 4 hours, the culture was then cultured at 20°C for 24-36 hours. After the culture was completed, the 96-deep-well plate was left to stand at 4°C for 3 hours, and then the supernatant was carefully discarded. The cells in the 96-deep-well plate were then divided into two portions, one of which was sealed and placed in a 50°C oven for 0.5 hours as a high-temperature treatment group, and the other was ultrasonically inactivated as a non-high-temperature treatment group. 20ul of the bacterial solution at the bottom of the deep-well plate was transferred to a new 96-well ELISA plate, and 280ul of the enzyme reaction solution (100mM potassium phosphate buffer, pH7.0, containing 100mM D-glucose, 0.6mMPMS, 0.12mM DCIP, 0.1% TritonX-100) was added, and the plate was incubated at 37°C on an ELISA reader for 5 minutes, and then the OD600 value was measured every 1 minute, with an interval of 10s between each measurement, and a total of 5-10 measurements were made. Finally, the residual enzyme activity of each mutant combination after 50°C treatment for 30 minutes relative to its non-high temperature treatment group was calculated based on the difference in OD600, and the enzyme activity without 50°C oven treatment for 0.5 hours was taken as 100%. The results are shown in Table 3.

[0076] Table 3 Stability scores of different mutation combinations calculated by BioPro software and residual enzyme activities after 50℃ treatment

[0077]

[0078]

[0079] According to the above thermal stability results, several plasmids (including blank plasmid pTrc99A) were selected for substrate specificity testing. The specific operation is: inoculate the selected mutant combination into a 50ml centrifuge tube at a density of 5%, add 15ml of autoinduction medium, and culture at 37°C until OD600>0.6, then transfer the centrifuge tube to a 20°C incubator and continue to culture for 24-36 hours. After the culture is completed, centrifuge at 7000rpm at room temperature for 10 minutes to collect the bacteria, and resuspend the bacteria with an equal volume of 100mM potassium phosphate buffer (containing 0.1% TritonX-100, pH7.0). After ultrasonicating the bacteria with an ultrasonicator, centrifuge at 14000rpm for 30 minutes and take the supernatant.

[0080] 20ul of supernatant was added to a 96-well ELISA plate pre-added with 30ul of sugar solution (1M glucose, 1M maltose, 1M galactose, 1M xylose, 1M fructose, 1M lactose, 1M sucrose) or water, and 250ul of enzyme reaction solution (100mM potassium phosphate buffer, pH7.0, containing 0.6mM PMS, 0.12mM DCIP, 0.1% TritonX-100) was added. The plate was incubated at 37°C on an ELISA reader for 5 minutes, and the OD600 value was measured every 1 minute. The interval between each measurement was 10s, and a total of 5-10 measurements were made. Finally, the reaction activity of each mutant combination for different sugars was calculated based on the difference in OD600. The activity of each mutant or enzyme in glucose was 100%. The results are shown in Tables 4 and 5.

[0081] Table 4 Response results of several mutation combinations to different sugars

[0082]

[0083]

[0084] Table 5 Enzyme activity results of several mutation combinations on different sugars

[0085]

[0086] Note: ND stands for not detectable.

[0087] It can be seen from the results in Tables 4 and 5 that the pTrc99A-6 mutant has better enzyme activity and specificity for glucose.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A glucose dehydrogenase mutant pTrc99A-6, characterized in that: The amino acid sequence of the glucose dehydrogenase mutant pTrc99A-6 is shown in SEQ ID NO.

1.

2. A nucleotide, characterized in that The nucleotide comprises a nucleotide sequence encoding the glucose dehydrogenase mutant pTrc99A-6 according to claim 1.

3. The nucleotide according to claim 2, characterized in that The nucleotide sequence is shown in SEQ ID NO.

2.

4. A carrier, characterized in that The vector carries the nucleotide according to claim 2 or 3.

5. The carrier according to claim 4, characterized in that The vector includes a plasmid.

6. A cell, characterized in that The cell carries the nucleotide according to claim 2 or 3, or contains the vector according to claim 4 or 5, or expresses the glucose dehydrogenase mutant pTrc99A-6 according to claim 1.

7. The cell according to claim 6, characterized in that Such cells include Escherichia coli and Pichia pastoris.

8. The method for preparing the glucose dehydrogenase mutant pTrc99A-6 according to claim 1, characterized in that: Obtained by fermentation of the cell according to claim 6 or 7.

9. Use of the glucose dehydrogenase mutant pTrc99A-6 according to claim 1 in the preparation of blood glucose detection products.

10. A blood sugar test strip, characterized in that: The glucose dehydrogenase method is used to detect blood sugar; the glucose dehydrogenase of the blood sugar test strip is the glucose dehydrogenase mutant pTrc99A-6 described in claim 1.

Images