Glucose dehydrogenase mutants, methods of making, uses and lyophilized powders

Abstract

The application provides a glucose dehydrogenase mutant, a preparation method, application and freeze-dried powder thereof, and relates to the technical field of biotechnology.The glucose dehydrogenase mutant provided by the application has an amino acid sequence as shown in SEQ ID NO.1.The glucose dehydrogenase mutant has better stability compared with a wild type, and has more specific 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, in particular to a glucose dehydrogenase mutant, a preparation method, application and freeze-dried powder 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, which can be used for blood glucose detection, and the detection is not interfered by general concentration of anticoagulants, preservatives, uric acid, bilirubin, etc. 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 the 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 its expression and separation and purification are very 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 it does not need oxygen to participate in the reaction process, 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 by the mainstream blood glucose meter on the market is FAD-dependent glucose dehydrogenase, which can be used from the 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, etc., 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, which has good stability and substrate specificity to solve the above technical problems.

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

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

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

[0010] A fifth object of the present application is to provide a method for preparing a glucose dehydrogenase mutant.

[0011] A sixth object of the present application is to provide a glucose dehydrogenase mutant lyophilized powder.

[0012] A seventh object of the present application is to provide the use of the above-mentioned glucose dehydrogenase mutant in the preparation of a blood glucose detection product.

[0013] To achieve the above objects, the following technical solutions are proposed:

[0014] In a first aspect, the present application provides a glucose dehydrogenase mutant, 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.

[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 above-mentioned 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 above-mentioned nucleotide, or contains the above-mentioned vector, or expresses the above-mentioned glucose dehydrogenase mutant.

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

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

[0022] In a sixth aspect, the present application provides a glucose dehydrogenase mutant lyophilized powder, which is prepared by mixing a glucose dehydrogenase mutant and a lyophilized liquid and then freeze-drying.

[0023] The freeze-dried liquid is an aqueous solution containing KPB, MgCl2 and sucrose, wherein the working concentration of KPB is 90-110 mM, the working concentration of MgCl2 is 0.18-0.22 mM, the mass concentration of sucrose is 4%-6%, and the pH is 6-8.

[0024] In a seventh aspect, the present application provides a use of the above-mentioned glucose dehydrogenase mutant in the preparation of a blood glucose detection product.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] The glucose dehydrogenase mutant provided by the present application has better stability and more specific substrate specificity than the wild type, and can be used to prepare a blood glucose detection product. BRIEF DESCRIPTION OF DRAWINGS

[0027] 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 prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0028] Figure 1 It is the color development result (10 minutes) of different monoclonal bacterial liquids on the enzyme-labeled plate;

[0029] Figure 2 It is the influence of methanol concentration on FAD-GDH expression amount;

[0030] Figure 3 It is the influence of different induction temperatures on FAD-GDH expression amount;

[0031] Figure 4 It is the influence of initial inoculum on FAD-GDH expression amount;

[0032] Figure 5 It is the influence of different induction pH on FAD-GDH expression amount;

[0033] Figure 6 It is the influence of different induction time on FAD-GDH expression amount;

[0034] Figure 7 It is the situation of pPIC9K-WT and pPIC9K-336-555 freeze-dried powder after reconstitution in 12% SDS-PAGE gel;

[0035] Figure 8 It is the standard curve of RD20230206-WT test paper for detecting blood glucose;

[0036] Figure 9 Standard curve for RD20230206-336-555 test paper to detect blood glucose. DETAILED DESCRIPTION

[0037] The embodiments of the present application will be described in detail below with reference to 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 of ordinary skill in the art without making creative efforts are within the scope of protection of the present application.

[0038] The term "vector" refers to a nucleic acid vehicle 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.

[0039] In a first aspect, the present application provides a glucose dehydrogenase mutant, the amino acid sequence of the glucose dehydrogenase mutant is shown as SEQ ID NO. 1:

[0040] TSAKYDYIVIGGGTSGLAVANRLSEDPSVNVLILEAGGSVWNNPNVTNVNGYGLAFGSDIDWQYQSVNQPYGGNVSQVLRAGKALGGTSTINGMAYTRAEDVQIDAWETIGNTGWTWKNLFPYYRKSENFTVPTKSQTSLGASYEAGAHGHEGPLDVAFTQIESNNLTTYLNRTFQGMGLPWTEDVNGGKMRGFNLYPSTVNLEEYVREDAARAYYWPYKSRPNLHVLLNTFANRIVWDGEARDGDITASGVEITSRNGTVRVINAEKEVIVSAGALKSPAILELSGIGNPSVLDKYNIPVKVNLPTVGENLQDQVNSHMDASGNTSISGTKAVSFPDVYDVFGDEAESVAKQIRANLKQYAADTAKANGNIMKAADLERLFEVQYDLIFKGRVPIAEVLNYPGSATSVFAEFWALLPFARGSVHIGSSNPAEFPVINPNYFMLDWDAKSYVAVAKYIRRSFESYPLSSIVKESTPGYDVIPRNASEQSWKEWVFDKNYRSNFHPVGTAAMMPREIGGVVDERLNVYGTTNVRVVDASVLPFQVCGHLVSTLYAMAERAADLIKADAGRR (SEQ ID NO. 1).

[0041] The inventors mutated the amino acids at positions 336 and 555 of wild-type glucose dehydrogenase and obtained a glucose dehydrogenase mutant. The inventors found that the mutant has better stability and more specific substrate specificity than the wild-type.

[0042] In a second aspect, the present application provides a nucleotide comprising a nucleotide sequence encoding the glucose dehydrogenase mutant.

[0043] The nucleotide can express the glucose dehydrogenase mutant of the present application.

[0044] In some optional embodiments, the nucleotide has a sequence as shown in SEQ ID NO. 2.

[0045]

[0046] In a third aspect, the present application provides a vector carrying the nucleotide.

[0047] The vector is introduced into a recipient cell, so that the recipient cell is capable of expressing the glucose dehydrogenase mutant of the present application.

[0048] In some alternative embodiments, the vector includes but is not limited to a plasmid.

[0049] In a fourth aspect, the present application provides a cell carrying the nucleotide, or containing the vector, or expressing the glucose dehydrogenase mutant.

[0050] 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.

[0051] In a fifth aspect, the present application provides a method for preparing the glucose dehydrogenase mutant, which is obtained by fermentation of the above-mentioned cell.

[0052] The preparation method is simple and efficient, and a large amount of glucose dehydrogenase mutant can be obtained by fermentation.

[0053] In a sixth aspect, the present application provides a glucose dehydrogenase mutant lyophilized powder, which is prepared by mixing the glucose dehydrogenase mutant and a lyophilized solution and then freeze-drying.

[0054] The lyophilized solution is an aqueous solution containing KPB, MgCl2 and sucrose, wherein the working concentration of KPB may be, but is not limited to, 90 mM, 100 mM or 110 mM, the working concentration of MgCl2 may be, but is not limited to, 0.18 mM, 0.2 mM or 0.22 mM, the mass concentration of sucrose may be, but is not limited to, 4%, 5% or 6%, and the pH may be, but is not limited to, 6, 7 or 8.

[0055] The inventors further optimize and adjust the lyophilized solution, so that the prepared glucose dehydrogenase mutant lyophilized powder is easy to store while retaining higher enzyme activity.

[0056] In a seventh aspect, the present application provides the use of the above-mentioned glucose dehydrogenase mutant in the preparation of blood glucose detection products.

[0057] The glucose dehydrogenase mutant provided by the present application has better stability than the wild type, and the substrate specificity is more specific, and can be used to prepare blood glucose detection products.

[0058] The present application is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are merely for the purpose of illustration and should not be construed as limiting the present application in any way.

[0059] Example 1

[0060] 1. Determination of glucose dehydrogenase mutants:

[0061] 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:

[0062] TSAKYDYIVIGGGTSGLAVANRLSEDPSVNVLILEAGGSVWNNPNVTNVNGYGLAFGSDIDW

[0063] QYQSVNQPYGGNVSQVLRAGKALGGTSTINGMAYTRAEDVQIDAWETIGNTGWTWKNLFPYYR

[0064] KSENFTVPTKSQTSLGASYEAGAHGHEGPLDVAFTQIESNNLTTYLNRTFQGMGLPWTEDVNGGK

[0065] MRGFNLYPSTVNLEEYVREDAARAYYWPYKSRPNLHVLLNTFANRIVWDGEARDGDITASGVEIT

[0066] SRNGTVRVINAEKEVIVSAGALKSPAILELSGIGNPSVLDKYNIPVKVNLPTVGENLQDQVNSHMD

[0067] ASGNTSISGTKAVSYPDVYDVFGDEAESVAKQIRANLKQYAADTAKANGNIMKAADLERLFEVQY

[0068] DLIFKGRVPIAEVLNYPGSATSVFAEFWALLPFARGSVHIGSSNPAEFPVINPNYFMLDWDAKSYVAV

[0069] AKYIRRSFESYPLSSIVKESTPGYDVIPRNASEQSWKEWVFDKNYRSNFHPVGTAAMMPREIGGVV

[0070] DERLNVYGTTNVRVVDASVLPFQVCGHLVSTLYAVAERAADLIKADAGRR (SEQ ID NO. 3).

[0071] 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 GOD active center, 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.

[0072] The selected mutation points are shown in Table 1, and the corresponding mutation primers are designed according to each mutation site.

[0073] Table 1 Selected mutation sites of FAD-GDH and mutation primers corresponding to the sites

[0074]

[0075]

[0076] The above 6 primers were synthesized by a primer synthesis company, and the mutation of the above sites was completed according to the instructions of the commercial mutation kit.

[0077] 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.

[0078] The above bacteria liquid was inoculated into a new 96-deep well plate at a density of 5%, and an appropriate amount of self-induction 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℃ for 4 hours and then at 20℃ for 24 hours, the 96-deep well plate was placed at 4℃ for 3 hours, and then the supernatant was carefully discarded. Then the 96-deep well plate was sealed and placed in a 50℃ oven for 0.5 hours. 20ul of bacteria liquid 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℃ 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 times. Finally, mutant colonies were screened according to the color of the enzyme-labeled plate and the difference in OD600 value, and the colorimetric results of the enzyme-labeled plate are shown in Figure 1

[0079] According to the color change results and the difference in OD600 value of the above enzyme-labeled plate, single clone strains were selected for sequencing by a sequencing company. The obtained sequencing results are shown in Table 2.

[0080] Table 2 Sequencing results of single clone strains for sequencing

[0081] 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

[0082] The 1 / 2 / 3 site mutations of the above sites were predicted by using the software BioPro, and the combination with a 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) plates and incubated at 37℃ overnight. After white colonies grew on the LB plates, 5 single colonies were selected from each plate for expansion culture, and incubated at 37℃ for 4-6 hours.

[0083] ​The above bacteria 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 inactivated by ultrasonic treatment as a non-high-temperature treatment group. 20ul of bacteria 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 50°C treatment for 30 minutes 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.

[0084] Table 3 Stability scores and residual enzyme activities of different mutant combinations at 50°C according to BioPro software

[0085]

[0086] According to the thermal stability results described above, several plasmids 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.

[0087] Take 20 ul supernatant added to pre-added 30 ul sugar solution (1M glucose, 1M maltose, 1M galactose, 1M xylose, 1M fructose, 1M lactose, 1M sucrose) or water in 96-well enzyme plate, add 250 ul enzyme reaction solution (100 mM potassium phosphate buffer, pH 7.0, containing 0.6 mM PMS, 0.12 mM DCIP, 0.1% Triton X-100), incubate in 37°C enzyme marker for 5 minutes, then measure OD600 value every 1 minute, 10s interval for each measurement, 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 sugars, each enzyme with its glucose activity as 100%, the results are shown in Table 4.

[0088] Table 4 reaction results of several mutant combinations on different sugars

[0089] Mutant name Glucose Maltose Galactose Xylose Fructose Lactose Sucrose pTrc99A-WT 100％ 7.2％ 2.0％ 8.2％ 0％ 0.6％ 0％ pTrc99A-2 100％ 16.8％ 1.6％ 7.8％ 0％ 1.0％ 0％ pTrc99A-4 100％ 15.3％ 2.4％ 4.8％ 0％ 2.1％ 0％ pTrc99A-5 100％ 12.9％ 1.2％ 7.1％ 2.0％ 0.5％ 0％ pTrc99A-6 100％ 8.1％ 0％ 4.8％ 0％ 0％ 0％ pTrc99A-7 100％ 18.3％ 2.4％ 17.0％ 1.9％ 2.0％ 0％ pTrc99A-9 100％ 6.0％ 0.9％ 3.6％ 0.2％ 0.2％ 0％ pTrc99A-15 100％ 4.3％ 1.9％ 2.2％ 1.2％ 1.2％ 0％

[0090] As can be seen from the results in Table 4, pTrc99A-15 mutant has good specificity for glucose.

[0091] 2. Expression and preparation of glucose dehydrogenase and its mutants:

[0092] Design primers, clone the target sequence on pTrc99A-15 and pTrc99A-WT into pPIC9K, add 8xHis tag at C terminal, enzyme cutting site is EcoRI / NotI, named as: pPIC9K-336-555 and pPIC9K-WT respectively.

[0093] Linearize the above plasmid with SacI single enzyme digestion, recover the product and transfer it into the competent cells of Pichia pastoris P. pastoris GS115 using an electroporator. After electroporation, the yeast cells are plated on MD plates and incubated at 30°C for 2-3 days until white colonies grow. Pick 6 single colonies from each plate that grow on MD plates into 30 ml of YPD medium (containing 1 mg / ml of G418) and incubate at 30°C, 250 rpm overnight.

[0094] Perform small-scale expression on the above single colonies (inoculate BMMY medium at a concentration of OD600=0.5, 30°C, 250 rpm, add 0.5% methanol every 24h, culture for 3 days), and select 1 highest expressing colony for each plasmid for subsequent optimization.

[0095] Table 5 expression of pPIC9K-WT or pPIC9K-336-555 in each colony

[0096] Clone number pPIC9K-WT (unit: U / ml) pPIC9K-336-555 (unit: U / ml) 1 20.1 18.7 2 18.9 16.5 3 16.0 19.9 4 21.0 21.0 5 25.5 17.4 6 19.9 17.6

[0097] According to the results of Table 5, the two selected single colonies pPIC9K-WT-5 and pPIC9K-336-555-4 were optimized for expression, and the specific experimental steps were as follows:

[0098] The strain was inoculated in BMY medium at a concentration of OD600=0.5, and cultured at pH 6.0, 30°C, 250 rpm, with the addition of different concentrations of methanol (0.5% vol, 1% vol, 1.5% vol, 2% vol, 2.5% vol) every 24 h, and cultured for 4 days, and the results are shown in Table 4. Figure 2

[0099] The strain was inoculated in BMY medium at a concentration of OD600=0.5, and cultured at pH 6.0, different temperatures (20°C, 25°C, 28°C, 30°C, 32°C), 250 rpm, with the addition of 1.5% vol of methanol every 24 h, and cultured for 4 days, and the results are shown in Table 5. Figure 3

[0100] The strain was inoculated in BMY medium at different concentrations (OD600=0.5, 1, 1.5, 5, 2.5), and cultured at pH 6.0, 28°C, 250 rpm, with the addition of 1.5% vol of methanol every 24 h, and cultured for 4 days, and the results are shown in Table 6. Figure 4

[0101] The strain was inoculated in BMY medium at a concentration of OD600=1.5, and cultured at different pH (5.5, 6, 6.5, 7, 7.5, 8), 28°C, 250 rpm, with the addition of 1.5% vol of methanol every 24 h, and cultured for 4 days, and the results are shown in Table 7. Figure 5

[0102] The strain was inoculated in BMY medium at a concentration of OD600=1.5, and cultured at pH 7.0, 28°C, 250 rpm, with the addition of 1.5% vol of methanol every 24 h, and the enzyme activity of the fermentation broth was collected every day, and the results are shown in Table 8. Figure 6

[0103] According to the results of Table 5, the two selected single colonies pPIC9K-WT-5 and pPIC9K-336-555-4 were optimized for expression, and the specific experimental steps were as follows: Figures 2-6 ​​​​​The optimal culture condition was obtained (inoculated at OD600=1.5 in BMMY medium with pH=7.0, 1.5% methanol was added every 24 h, 28 °C, 250 rpm for 7 days) and under the optimal culture condition, pPIC9K-WT and pPIC9K-336-555 were each fermented in a flask for 5 L, 8000 rpm, and the supernatant was collected by centrifugation. The supernatant was passed through a 30 KDa tangential flow, and concentrated to 300 ml. Purification was performed using a Ni-excel column, with a 50 mM sodium phosphate buffer, pH 7.0 (15 mM imidazole for washing, 500 mM imidazole for elution). The purified enzyme solution obtained by purification was dialyzed into 100 mM potassium phosphate buffer (pH 7.0).

[0104] 3. Preparation of glucose dehydrogenase and mutant thereof freeze-dried powder:

[0105] The freeze-drying formula was explored using the purified enzyme obtained by pPIC9K-WT purification, and the following freeze-drying liquid was configured at a FAD-GDH concentration of 1 mg / ml (Table 6). 1 ml of freeze-drying liquid was added to a 5 ml vial and placed at -80 °C for 1 hour, then quickly placed in a pre-cooled freeze dryer, and the freeze-drying experiment was performed according to the program.

[0106] The enzyme activity of each formula was determined in the same way as the enzyme activity determination, and the freeze-drying liquid No. 0 placed at 4 °C was taken as 100% enzyme activity. The protection effect of each freeze-drying liquid formula is shown in Table 6.

[0107] Table 6 Protection of different freeze-drying liquid formulas on FAD-GDH

[0108]

[0109]

[0110] As can be seen from the results in Table 6, No. 4 freeze-drying liquid is the best freeze-drying night formula. According to the No. 4 freeze-drying night formula, the enzyme solution of pPIC9K-WT and pPIC9K-336-555 was made into freeze-dried powder, and the specific enzyme activity of the two before and after freeze-drying was detected, and the results are shown in Table 7.

[0111] Table 7 Specific enzyme activity of pPIC9K-WT and pPIC9K-336-555 before and after freeze-drying

[0112] Name Before freeze-drying (Bradford specific enzyme activity) After freeze-drying (weighed specific enzyme activity) pPIC9K-WT 900 U / mg 600 U / mg pPIC9K-336-555 550 U / mg 380 U / mg

[0113] After reconstituting the freeze-dried powder, electrophoresis detection was performed, and the protein bands of pPIC9K-WT and pPIC9K-336-555 are shown in Table 7. Figure 7

[0114] ​4. Performance verification of glucose dehydrogenase and its mutant lyophilized powder in blood glucose detection:

[0115] After the above lyophilized powder is compounded into a related solution, it is dried to prepare a blood glucose monitoring test paper. The test is as follows:

[0116] 1) Linear correlation

[0117] Experimental materials:

[0118] Blood (No. E83922263, E83922498; initial Hct: 43.6%); Acutek blood glucose meter; test paper batch number: RD20230206-WT (lyophilized enzyme), RD20230206-336-555 (lyophilized enzyme).

[0119] Experimental steps:

[0120] The above blood was used to dilute glucose according to the target concentration, and the blood glucose target concentration (mg / dL) was 0, 54, 110, 180, 275, 360, 450, 525, 615, 800. After dilution, two groups of samples YSI 1 and YSI 2 were obtained, and then the same concentration of YSI 1 and YSI 2 were mixed in equal volume, and after cell removal, the plasma sample Plasma YSI was obtained.

[0121] Each blood sample tested 10 reagent strips for each batch of blood glucose meter.

[0122] Experimental data:

[0123] The current value was detected for 5s, and the results are shown in Tables 8-9 and Figures 8-9 The overall evaluation of the dehydrogenase batch WT, 336-555 linear correlation is good.

[0124] Table 8

[0125]

[0126]

[0127] Note: YSI 1: actual concentration of glucose in YSI 1 group sample; YSI 2: actual concentration of glucose in YSI 2 group sample; Avg (mg / dL): average concentration of YSI 1 and YSI 2; Plasma YSI: concentration of glucose in plasma sample; Rep. 1-10: current value detected by test paper No. 1-10; Avg (uA): average current value of 10 test papers (if not specified, the meaning of the first column in the following table is the same as that in Table 8).

[0128] Table 9

[0129]

[0130] 2) Substrate specificity

[0131] Experimental materials:

[0132] Blood number: B19394773, B19394427; Initial Hct: 37.6%;

[0133] Blood glucose meter: Acu-Check blood glucose meter

[0134] Test strip batch number: RD20230206-WT (lyophilized enzyme), RD20230206-336-555 (lyophilized enzyme)

[0135] Experimental procedure:

[0136] The above blood was used to dilute glucose and fructose, lactose, galactose or maltose according to the target concentration or test concentration, respectively. The target concentration of glucose (mg / dL) was 110 and 350, the test concentration of fructose (mg / dL) was 30 and 100, the test concentration of lactose (mg / dL) was 5 and 25, the test concentration of galactose (mg / dL) was 78 and 100, and the test concentration of maltose (mg / dL) was 40 and 100. After dilution, two groups of samples YSI 1 and YSI 2 were obtained. Then the same concentration of YSI 1 and YSI 2 were mixed in equal volume to obtain the plasma sample Plasma YSI.

[0137] Each blood sample was tested with 10 reagent strips per batch number.

[0138] Experimental data:

[0139] The current value was detected at 5s, and the results are shown in Tables 10-11.

[0140] Table 10

[0141]

[0142] Table 11

[0143]

[0144]

[0145] Overall, RD20230818-WT and RD20230818-336-555 were not affected by fructose, lactose, and galactose, but RD20230818-WT had poor individual value accuracy and was at risk of being affected by maltose.

[0146] 3) Accelerated stability

[0147] Experimental materials:

[0148] Blood number: 44238432, 44238495; Initial Hct: 38.4%.

[0149] Test fixture: Current host computer;

[0150] Test strip batch number: RD20230818-WT, RD20230818-336-555.

[0151] Experimental steps:

[0152] The blood was diluted with glucose according to the target concentration, and the target concentration of blood glucose (mg / dL) was 50, 110, 275, and 525. After dilution, two groups of samples YSI 1 and YSI 2 were obtained. Then the same concentration of YSI 1 and YSI 2 was mixed in equal volume to obtain the plasma sample Plasma YSI.

[0153] Five reagent strips were tested for each blood sample and each batch number by the current host computer.

[0154] Experimental data:

[0155] The current value was detected for 5s, and the results are shown in Tables 12-13. Overall evaluation of the accelerated stability results of RD20230818-WT does not meet the two-year storage requirement at high values, and the accelerated stability results of RD20230818-336-555 meet the two-year storage requirement.

[0156] Table 12

[0157]

[0158] Note: "65℃ 1W" means 1 week at 65℃, and the same applies to RT (2 weeks at room temperature); Bias vs. RT refers to the deviation of the measured current value from RT (room temperature).

[0159] Table 13

[0160]

[0161]

[0162] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, 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, characterized in that, The amino acid sequence of the glucose dehydrogenase mutant is shown in SEQ ID NO.

1.

2. A nucleotide, characterized in that, The nucleotides include the nucleotide sequence encoding the glucose dehydrogenase mutant 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 includes plasmids.

6. A cell, characterized in that, The cell carries the nucleotide of claim 2 or 3, or contains the vector of claim 4 or 5, or expresses the glucose dehydrogenase mutant of claim 1; The cells in question are not plant or animal varieties.

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

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

9. A glucose dehydrogenase mutant lyophilized powder, characterized in that, It is prepared by mixing the glucose dehydrogenase mutant according to claim 1 with a lyophilized solution and then freeze-drying. The freeze-drying solution is an aqueous solution containing KPB, MgCl2 and sucrose, wherein the working concentration of KPB is 90-110 mM, the working concentration of MgCl2 is 0.18-0.22 mM, the mass concentration of sucrose is 4%-6%, and the pH is 6-8.

10. The application of the glucose dehydrogenase mutant according to claim 1 in the preparation of blood glucose detection products.

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