Glucose dehydrogenase mutant pTrc99a-6, preparation method, application and blood glucose detection test paper thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly to a glucose dehydrogenase mutant pTrc99A-6, a preparation method, application and blood glucose test paper thereof. BACKGROUND

[0002] Glucose dehydrogenase (GDH) is an oxidoreductase that can catalyze the dehydrogenation and oxidation of glucose to generate gluconolactone. In the reaction process, glucose loses electrons and transfers to the coenzyme or prosthetic group of GDH to generate reduced prosthetic group or coenzyme, and the content of the generated reduced prosthetic group or coenzyme is positively correlated with the concentration of glucose. Accordingly, the glucose detection can be used, and the detection is not interfered by general concentration of anticoagulants, preservatives, uric acid, bilirubin and the like. Compared with the traditional GOD method and HK method, the GDH method is more simple to operate, only one enzyme is needed to participate in the reaction, and both continuous monitoring (λ = 340 nm) and end-point analysis can be realized, so it is often used for blood glucose determination.

[0003] GDH can be divided into PQQ-dependent GDH (PQQ-GDH), NAD-dependent GDH (NAD-GDH) and FAD-dependent GDH (FAD-GDH) according to different bound prosthetic groups or coenzymes. The substrate specificity of soluble sPQQ-GDH is poor, and the substrate spectrum is too wide; the mPQQ-GDH of membrane-bound type is a membrane protein, and the expression and separation and purification are difficult, which seriously limits its application in blood glucose determination. NAD-GDH needs to continuously add coenzyme to maintain its activity due to the loose coenzyme binding, which is also not conducive to blood glucose detection. FAD-GDH has tight coenzyme binding and is not easy to fall off, and the reaction process does not need the participation of oxygen, so it is superior to glucose oxidase and other coenzyme-dependent GDH in terms of detection accuracy and long-term preservation. The core enzyme used in the mainstream blood glucose meter on the market is FAD-dependent glucose dehydrogenase, which can be used from opening to the end of the effective period.

[0004] Wild-type FAD-GDH from different sources mostly has multiple substrate activities, and still has certain dehydrogenation and oxidation activity on maltose and xylose, and the specificity is poor. If such components exist in blood, false blood glucose will occur when detecting blood glucose.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] The first object of the present application 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 application is to provide a nucleotide.

[0008] The third object of the present application is to provide a vector.

[0009] The fourth object of the present application is to provide a cell.

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

[0011] The sixth object of the present application is to provide an application of the glucose dehydrogenase mutant pTrc99A-6 in preparing a blood glucose detection product.

[0012] The seventh object of the present application is to provide a blood glucose detection test paper.

[0013] In order to achieve the above objects, the following technical solutions are proposed.

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

[0015] In a second aspect, the present application provides a nucleotide, which comprises 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 in SEQ ID NO. 2.

[0017] In a third aspect, the present application provides a vector, which carries the nucleotide.

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

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

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

[0021] In a fifth aspect, the present application provides a preparation method of the glucose dehydrogenase mutant pTrc99A-6, which is obtained by fermentation using the above-mentioned cell.

[0022] In a sixth aspect, the present application provides an application of the above-mentioned glucose dehydrogenase mutant pTrc99A-6 in preparing a blood glucose detection product.

[0023] In a seventh aspect, the present application provides a blood glucose test paper for detecting blood glucose by using glucose dehydrogenase method; the glucose dehydrogenase of the blood glucose test paper is the glucose dehydrogenase mutant pTrc99A-6.

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

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

[0026] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0027] Figure 1 Color development results (10 minutes) of different monoclonal bacterial liquids on the enzyme-labeled plate. DETAILED DESCRIPTION

[0028] The embodiments of the present application will be described in detail below in combination 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 application and should not be regarded as limiting the scope of the present application. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0029] The term "vector" refers to a nucleic acid carrier into which a nucleotide can be inserted. When the vector can make the protein encoded by the inserted polynucleotide to be expressed, 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 carried by the vector can be expressed in the host cell.

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

[0031] TSAKYDYIVIGGGTSGLAVANRLSEDPSVNVLILEAGGSVWNNPNVTNVNGYGLAFGSDIDWQYQSVNQPYGGNVSQVLRAGKALGGTSTINGMAYTRAEDVQIDAWETIGNTGWTWKNLFPYYRKSENFTVPTKSQTSLGASYEAGAHGHEGPLDVAFTQIESNNLTTYLNRTFQGMGLPWTEDVNGGKMRGFNLYPSTVNLEEYVREDAARAYYWPYKSRPNLHVLLNTFANRIVWDGEARDGDITASGVEITSRNGTVRVINAEKEVIVSAGALKSPAILELSGIGNPSVLDKYNIPVKVNLPTVGENLQDQVNSHMDASGNTSISGTKAVSFPDVYDVFGDEAESVAKQIRANLKQYAADTAKANGNIMKAADLERLFEVQYDLIFKGRVPIAEVLNYPGSATSVFAEFWALLPFARGSVHIGSSNPAEFPVINPNYFMLDWDAKSYVAVAKYIRRSFESYPLSSIVKESTPGYDVIPRNASEQSWKEWVFDKNYRSNFHPVGTAAMMPREIGGVVDERLNVYGTTNVRVVDASVLPFQVCGHLVSTLYAYAERAADLIKADAGRR (SEQ ID NO. 1).

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

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

[0034] The nucleotide can express the glucose dehydrogenase mutant pTrc99A-6 of the present application.

[0035] In some optional embodiments, the nucleotide has a sequence as shown in SEQ ID NO. 2.

[0036]

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

[0038] The vector is introduced into a recipient cell, so that the recipient cell is capable of expressing the glucose dehydrogenase mutant pTrc99A-6.

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

[0040] In a fourth aspect, the present application provides a cell carrying the nucleotide, or containing the vector, or expressing the glucose dehydrogenase mutant pTrc99A-6.

[0041] In some alternative embodiments, the cell includes but is not limited to Escherichia coli and Pichia pastoris, and other cells known to those skilled in the art.

[0042] In a fifth aspect, the present application provides a preparation method of the glucose dehydrogenase mutant pTrc99A-6, which is obtained by fermentation of the above-mentioned cell.

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

[0044] In a sixth aspect, the present application 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 application has better stability and more specific substrate specificity than the wild type, and can be used to prepare blood glucose detection products.

[0046] In a seventh aspect, the present application provides a blood glucose detection test paper for detecting blood glucose by glucose dehydrogenase method; the glucose dehydrogenase of the blood glucose detection test paper is the glucose dehydrogenase mutant pTrc99A-6.

[0047] Since the glucose dehydrogenase mutant pTrc99A-6 has better specificity, the blood glucose detection test paper prepared by the mutant has higher accuracy.

[0048] The present application will be further described below by specific examples and comparative examples, but it should be understood that these examples are only for more detailed description and should not be understood as limiting the present application in any form.

[0049] Example 1

[0050] 1. Determination of glucose dehydrogenase mutant:

[0051] The FAD-GDH original sequence plasmid selected in this study is named 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, first, the GOD crystal structure 1CF3 downloaded in the PDB database was compared with wild-type FAD-GDH using the software PyMOL, the active center residues of FAD-GDH were found according to the conserved residues of the active center of GOD, and the differences in the structures around the active sites of the two structures were compared. According to the structure comparison of FAD-GDH and GOD, the Autodock software was used for molecular docking to obtain the substrate binding pocket model of FAD-GDH and D-glucose, and the interaction between the enzyme and the substrate was analyzed. According to the model and the results of the previous structure comparison, the mutation sites were determined.

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

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

[0067]

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

[0069] After the above mutations were completed, the obtained plasmids were transformed into E. coli DH5α competent cells, spread on LB (containing Amp resistance) plates, and cultured at 37°C overnight. After white colonies grew on the LB plate, the above colonies were picked into a 96-deep well plate for expansion culture, and cultured at 37°C for 4-6 hours.

[0070] The above bacteria 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, 1x trace element, 10 mM potassium phosphate buffer, pH 7.0) was added. After incubation at 37°C for 4 hours and then at 20°C for 24 hours, the 96-deep well plate was placed at 4°C for 3 hours, and then the supernatant was carefully discarded. Then the 96-deep well plate was sealed and placed in a 50°C oven for 0.5 hours. 20ul of bacteria solution at the bottom of the deep well plate was transferred to a new 96-well enzyme-labeled plate, 280ul of reaction solution (100 mM potassium phosphate buffer, pH 7.0, containing 100 mM D-glucose, 0.6 mM PMS (phenazine methosulfate), 0.12 mM DCIP (2,6-dichloroindophenol sodium), 0.1% Triton X-100) was added, and the enzyme-labeled plate was incubated at 37°C for 5 minutes. Then the OD600 value was measured every 1 minute, with a 10s interval between each measurement, for a total of 5-10 measurements. Finally, the mutant colonies were screened according to the color change of the enzyme-labeled plate and the difference in OD600 value, and the color change result of the enzyme-labeled plate is shown in Figure 1

[0071] According to the color change result of the enzyme-labeled plate and the difference in OD600 value, the single clone strain was selected for sequencing by a sequencing company. The obtained sequencing result is shown in Table 2.

[0072] Table 2 Sequencing results of single clone strains 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] The 1 / 2 / 3 site mutation of the above site was predicted by using the software BioPro, and the combination with higher score was selected for synthesis by a company. After obtaining the plasmid, the plasmid was transformed into competent cells of Escherichia coli BL21 (DE3), and then coated on LB (containing Amp resistance) plate and incubated at 37°C overnight. After white colonies grew on the LB plate, 5 single colonies were selected from each plate for expansion culture, and incubated 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 incubation at 37°C for 4 hours and at 20°C for 24-36 hours, the 96-deep well plate was placed 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 parts, 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 bacterial solution at the bottom of the deep well plate was transferred to a new 96-well enzyme-labeled plate, 280ul of enzyme reaction solution (100mM potassium phosphate buffer, pH7.0, containing 100mM D-glucose, 0.6mM PMS, 0.12mM DCIP, 0.1% TritonX-100) was added, and the enzyme-labeled plate was incubated at 37°C for 5 minutes. Then the OD600 value was measured every 1 minute, with a 10s interval between each measurement, for a total of 5-10 measurements. Finally, the residual enzyme activity of each mutant combination after 30 minutes of 50°C treatment was calculated relative to its non-high-temperature treatment group, with the enzyme activity of the non-50°C oven treatment group for 0.5 hours as 100%, and the results are shown in Table 3.

[0076] Table 3 Stability scores and residual enzyme activities at 50°C of different mutant combinations calculated according to BioPro software

[0077]

[0078]

[0079] According to the thermal stability results described above, several plasmids (including the blank plasmid pTrc99A) were selected for substrate specificity testing. Specifically, the selected mutant combinations were inoculated into 50ml centrifuge tubes at a density of 5%, and 15ml of autoinduction medium was added. After incubation at 37°C until OD600>0.6, the centrifuge tubes were transferred to a 20°C incubator for further incubation for 24-36 hours. After incubation, the bacterial cells were collected by centrifugation at 7000rpm for 10 minutes at room temperature, and the bacterial cells were resuspended with an equal volume of 100mM potassium phosphate buffer (containing 0.1% TritonX-100, pH7.0). After ultrasonic treatment of the bacterial cells, the supernatant was obtained by centrifugation at 14000rpm for 30 minutes.

[0080] Take 20ul supernatant added in pre-added 30ul sugar solution (1M glucose, 1M maltose, 1M galactose, 1M xylose, 1M fructose, 1M lactose, 1M sucrose) or water in 96-well enzyme plate, add 250ul enzyme reaction solution (100mM potassium phosphate buffer, pH7.0, containing 0.6mM PMS, 0.12mM DCIP, 0.1% TritonX-100), incubate in 37℃ enzyme reader for 5 minutes, then measure OD600 value every 1 minute, each measurement interval shake 10s, a total of 5-10 times, finally according to the difference of OD600 value to calculate the reaction activity of each mutant combination for different sugar, each mutant or enzyme is 100% of its glucose activity, the results are shown in table 4 and table 5.

[0081] Table 4 reaction results of several mutant combinations on different sugars

[0082]

[0083]

[0084] Table 5 enzyme activity results of several mutant combinations on different sugars

[0085]

[0086] Note: ND is not detected.

[0087] From the results of table 4 and table 5, it can be seen that pTrc99A-6 mutant has better enzyme activity and specificity for glucose.

[0088] Finally, it should be noted that: the above examples are used to illustrate the technical solutions of the present application, but not limited to them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

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 sequence is the nucleotide sequence encoding the glucose dehydrogenase mutant pTrc99A-6 of claim 1.

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

2.

4. A carrier, characterized in that, The carrier carries the nucleotides as described in claim 2 or 3.

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

6. A cell, characterized in that, The cell carries the nucleotides of claim 2 or 3, or contains the vector of claim 4 or 5, or expresses the glucose dehydrogenase mutant pTrc99A-6 of claim 1; the cell is a non-plant cell.

7. The cell according to claim 6, characterized in that, The cells are either Escherichia coli or Pichia pastoris.

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

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

10. A blood glucose test strip, characterized in that, Blood glucose was detected using the glucose dehydrogenase method; the glucose dehydrogenase in the blood glucose test strip was the glucose dehydrogenase mutant pTrc99A-6 as described in claim 1.

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