Glucose dehydrogenase mutant pTrc99A-7 as well as preparation method and application thereof

Abstract

The invention provides a glucose dehydrogenase mutant pTrc99A-7 as well as a preparation method and application thereof, and relates to the technical field of biology. The amino acid sequence of the glucose dehydrogenase mutant pTrc99A-7 provided by the invention is as shown in SEQ ID NO. 1. The glucose dehydrogenase mutant pTrc99A-7 is good in thermal stability, has catalytic activity of various substrates, is wide in pH tolerance range, and is beneficial to solving the problems of single catalytic substrate, low utilization rate, single use mode, limited application range and the like of glucose dehydrogenase.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a glucose dehydrogenase mutant pTrc99A-7 and a preparation method and application thereof. Background Art

[0002] FAD-GDH (flavin adenine dinucleotide-dependent glucose dehydrogenase, EC 1.1.5.9) is a type of oxidoreductase that catalyzes the oxidation of glucose to produce gluconolactone, with flavin adenine dinucleotide (FAD) as its coenzyme. It is mainly derived from filamentous fungi. So far, the FAD-GDH that has been discovered and identified mainly comes from Aspergillus oryzae, Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Aspergillus carbonii, Penicillium and Cunninghamia lanceolata.

[0003] However, the FAD-GDH produced by these wild strains has problems such as low enzyme activity, sensitivity to solution pH, poor tolerance, narrow range of application, and poor thermal stability, which limits its application in clinical testing, industrial applications, test paper testing, preparation of biosensors, regeneration of coenzymes, development of new fuel cells and pacemakers, and limits the scope of industry application.

[0004] In addition, existing modifications to FAD-GDH are mostly aimed at improving its specificity for glucose catalysis, resulting in problems such as a single catalytic substrate for the enzyme, low utilization rate, a single method of use, a limited scope of application, an inability to break through the existing model in industry applications, and an inability to form linkages between industries.

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

[0006] The first purpose of the present invention is to provide a glucose dehydrogenase (FAD-GDH) mutant pTrc99A-7, 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-7.

[0011] The sixth object of the present invention is to provide the use of the above-mentioned glucose dehydrogenase mutant pTrc99A-7 in the catalysis of glucose, maltose, galactose, xylose, fructose or lactose.

[0012] In order to achieve the above objectives, the following technical solutions are proposed:

[0013] In a first aspect, the present invention provides a glucose dehydrogenase mutant pTrc99A-7, the amino acid sequence of the glucose dehydrogenase mutant pTrc99A-7 is shown in SEQ ID NO.1.

[0014] In a second aspect, the present invention provides a nucleotide comprising a nucleotide sequence encoding the above-mentioned glucose dehydrogenase mutant pTrc99A-7.

[0015] As a further technical solution, the sequence of the nucleotide is shown as SEQ ID NO.2.

[0016] In a third aspect, the present invention provides a vector carrying the nucleotide.

[0017] As a further technical solution, the vector includes a plasmid.

[0018] In a fourth aspect, the present invention provides a cell, which carries the nucleotide, or contains the vector, or expresses the glucose dehydrogenase mutant pTrc99A-7.

[0019] As a further technical solution, the cells include Escherichia coli and Pichia pastoris.

[0020] In a fifth aspect, the present invention provides a method for preparing a glucose dehydrogenase mutant pTrc99A-7, which is obtained by fermentation of the above-mentioned cells.

[0021] As a further technical solution, the fermentation conditions include: the initial inoculum amount is OD600=0.5-1.5, the pH is 6.5-7.5, the temperature is 25-30°C, and 1.5% methanol of the culture medium volume is added every 24 hours.

[0022] In a sixth aspect, the present invention provides the use of the glucose dehydrogenase mutant pTrc99A-7 in the catalysis of glucose, maltose, galactose, xylose, fructose or lactose.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The glucose dehydrogenase mutant pTrc99A-7 provided by the present invention has good thermal stability, catalytic activity on multiple substrates, and a wide pH tolerance range, which helps to solve the problems of single catalytic substrate, low utilization rate, single usage mode, and limited application range of glucose dehydrogenase. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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.

[0026] Figure 1 The color development results of different monoclonal bacterial solutions on the ELISA plate (10 minutes);

[0027] Figure 2 is the effect of methanol concentration on the expression of FAD-GDH;

[0028] Figure 3 is the effect of induction temperature on the expression of FAD-GDH;

[0029] Figure 4 is the effect of the initial inoculum size on the expression of FAD-GDH;

[0030] Figure 5 To show the effect of induced pH on the expression of FAD-GDH;

[0031] Figure 6 is the effect of induction time on the expression of FAD-GDH;

[0032] Figure 7 This is the temperature stability result of FAD-GDH;

[0033] Figure 8 This is the pH stability result of FAD-GDH. DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] In a first aspect, the present invention provides a glucose dehydrogenase mutant pTrc99A-7, the amino acid sequence of the glucose dehydrogenase mutant pTrc99A-7 is shown in SEQ ID NO.1:

[0037] (SEQ ID NO.1).

[0038] The inventor mutated the amino acids at positions 118 and 160 of wild-type glucose dehydrogenase and obtained the glucose dehydrogenase mutant pTrc99A-7. The inventor found that the mutant has good thermal stability, catalytic activity with multiple substrates, and a wide pH tolerance range, which helps to solve the problems of single catalytic substrates, low utilization rate, single usage mode, and limited application range of glucose dehydrogenase; it is conducive to improving its application in clinical testing, industrial application, test paper testing, preparation of biosensors, regeneration of coenzymes, development of new fuel cells and pacemakers, and thus breaking through the existing fixed production and application mode in the industry field, promoting linkage between industries, and facilitating the integration and optimization of scientific and technological resource allocation and improving resource allocation efficiency.

[0039] In a second aspect, the present invention provides a nucleotide comprising a nucleotide sequence encoding the above-mentioned glucose dehydrogenase mutant pTrc99A-7.

[0040] The nucleotide can be expressed and translated to obtain the glucose dehydrogenase mutant pTrc99A-7 of the present invention.

[0041] In some optional embodiments, the nucleotide sequence is shown in SEQ ID NO.2:

[0042]

[0043] In a third aspect, the present invention provides a vector carrying the nucleotide.

[0044] By introducing the vector into the recipient cells, the recipient cells can express the glucose dehydrogenase mutant pTrc99A-7 of the present invention.

[0045] In some optional embodiments, the vector includes but is not limited to a plasmid.

[0046] In a fourth aspect, the present invention provides a cell, which carries the nucleotide, or contains the vector, or expresses the glucose dehydrogenase mutant pTrc99A-7.

[0047] 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.

[0048] In a fifth aspect, the present invention provides a method for preparing a glucose dehydrogenase mutant pTrc99A-7, which is obtained by fermentation of the above-mentioned cells.

[0049] The preparation method is simple and efficient, and a large amount of glucose dehydrogenase mutant pTrc99A-7 can be obtained through fermentation.

[0050] In some optional embodiments, the fermentation conditions include: an initial inoculum amount of OD600 = 0.5-1.5, a pH of 6.5-7.5, a temperature of 25-30°C, and adding 1.5% methanol by volume of the culture medium every 24 hours.

[0051] In a sixth aspect, the present invention provides the use of the glucose dehydrogenase mutant pTrc99A-7 in the catalysis of glucose, maltose, galactose, xylose, fructose or lactose.

[0052] The glucose dehydrogenase mutant pTrc99A-7 provided by the invention has catalytic activity to glucose, maltose, galactose, xylose, fructose and lactose, and thus can be used for the catalysis of glucose, maltose, galactose, xylose, fructose and lactose.

[0053] 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.

[0054] Example 1

[0055] 1. Determination of glucose dehydrogenase mutants:

[0056] 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:

[0057] TSAKYDYIVIGGGTSGLAVANRLSEDPSVNVLILEAGGSVWNNPNV

[0058] TNVNGYGLAFGSDIDWQYQSVNQPYGGNVSQVLRAGKALGGTSTING

[0059] MAYTRAEDVQIDAWETIGNTGWTWKNLFPYYRKSENFTVPTKSQTSLG

[0060] ASYEAGAHGHEGPLDVAFTQIESNNLTTYLNRTFQGMGLPWTEDVNGG

[0061] KMRGFNLYPSTVNLEEYVREDAARAYYWPYKSRPNLHVLLNTFANRIV

[0062] WDGEARDGDITASGVEITSRNGTVRVINAEKEVIVSAGALKSPAILELSGI

[0063] GNPSVLDKYNIPVKVNLPTVGENLQDQVNSHMDASGNTSISGTKAVSYP

[0064] DVYDVFGDEAESVAKQIRANLKQYAADTAKANGNIMKAADLERLFEVQ

[0065] YDLIFKGRVPIAEVLNYPGSATSVFAEFWALLPFARGSVHIGSSNPAEFPVI

[0066] NPNYFMLDWDAKSYVAVAKYIRRSFESYPLSSIVKESTPGYDVIPRNASE

[0067] QSWKEWVFDKNYRSNFHPVGTAAMMPREIGGVVDERLNVYGTTNVRV

[0068] VDASVLPFQVCGHLVSTLYAVAERAADLIKADAGRR(SEQ ID NO.3)。

[0069] 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.

[0070] The selected mutation points are shown in Table 1, and corresponding mutation primers were designed according to each mutation site.

[0071] Table 1 Selected mutation sites of FAD-GDH and corresponding mutation primers

[0072]

[0073] The above 6 primers were synthesized by a primer synthesis company, and the mutation of the above sites was completed using a commercial mutagenesis kit according to the instructions.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] Table 2 Summary of sequencing results of monoclonal strains sent for sequencing

[0078] 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

[0079] 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.

[0080] 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.

[0081] Table 3 Stability scores of different mutation combinations calculated by BioPro software and residual enzyme activities after 50℃ treatment

[0082]

[0083]

[0084] 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.

[0085] 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 enzyme in glucose was 100%. The results are shown in Table 4.

[0086] Table 4 Response results of several mutation combinations to different sugars

[0087]

[0088]

[0089] From the results in Table 4, it can be seen that, in addition to glucose, the catalytic activity of the pTrc99A-7 mutant on maltose, xylose, fructose and lactose is significantly improved compared with the wild-type strain.

[0090] 2. Expression and preparation of glucose dehydrogenase mutant, pTrc99A-7:

[0091] Primers were designed to clone the target sequences on pTrc99A-7 and pTrc99A-WT into pPIC9K, 8xHistag was added to the C-terminus, and the restriction sites were EcoRI / NotI. They were named: pPIC9K-118-160 and pPIC9K-WT, respectively.

[0092] The plasmid was linearized by single enzyme digestion with SacI, and the recovered product was transferred into competent cells of Pichia pastoris GS115 using an electroporator. After electroporation, the yeast cells were spread on MD plates and cultured at 30°C for 2-3 days until white colonies grew. Six single colonies grown on each MD plate were picked and placed in 30 ml YPD medium (containing 1 mg / ml G418) and cultured overnight at 30°C and 250 rpm.

[0093] The above single colonies were expressed in small quantities (inoculated in BMMY medium at a concentration of OD600=0.5, 30°C, 250rpm, 0.5% methanol was added every 24h, and cultured for 3 days), and one colony with the highest expression was selected for each plasmid for subsequent optimization.

[0094] Table 5 Expression of pPIC9K-WT or pPIC9K-118-160 in each colony

[0095] Clone pPIC9K-WT pPIC9K-118-160 1 20.1 16.8 2 18.9 17.2 3 16.0 22.3 4 21.0 20.6 5 25.5 15.6 6 19.9 18.4

[0096] Based on the above results, the expression levels of the two monoclonal colonies, pPIC9K-WT-5 and pPIC9K-118-160-3, were optimized. The specific experimental steps are as follows:

[0097] The strain was inoculated into BMY medium at a concentration of OD600 = 0.5 and cultured at pH 6.0, 30°C, and 250 rpm. Different concentrations of methanol (0.5% vol, 1% vol, 1.5% vol, 2% vol, and 2.5% vol) were added every 24 h. The culture was carried out for 4 days. The results are as follows: Figure 2 As shown;

[0098] The strain was inoculated into 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), 250rpm, and 1.5% vol methanol was added every 24h for 4 days. The results are as follows Figure 3 As shown;

[0099] The strain was inoculated into BMY medium at different concentrations (OD600 = 0.5, 1, 1.5, 5, 2.5), cultured at pH 6.0, 28°C, 250 rpm, and 1.5% vol methanol was added every 24 h for 4 days. The results are as follows: Figure 4 As shown;

[0100] The strain was inoculated into 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, 250rpm, and 1.5% vol methanol was added every 24h for 4 days. The results are as follows Figure 5 As shown;

[0101] The strain was inoculated into BMY medium at a concentration of OD600 = 1.5, and cultured at pH 7.0, 28°C, and 250 rpm. 1.5% vol methanol was added every 24 h, and the fermentation broth was collected every day to measure the enzyme activity. The results are as follows: Figure 6 shown.

[0102] According to the above optimal culture scheme (inoculated in BMMY medium with pH = 7.0 at a concentration of OD600 = 1.5, adding 1.5% methanol every 24 hours, 28°C, 250rpm continuous culture for 7 days), pPIC9K-WT and pPIC9K-118-160-3 were fermented in 5L shake flasks, 8000rpm, and the supernatant was collected by centrifugation. The supernatant was concentrated to 300ml by 30KDa tangential flow. Purified with Ni-excel column, the buffer was 50mM sodium phosphate buffer, pH7.0 (15mM imidazole washing, 500mM imidazole elution). The purified pure enzyme solution was dialyzed into 100mM potassium phosphate buffer (pH7.0).

[0103] 3. Thermal stability test of enzyme solution obtained from pPIC9K-118-160-3 mutant and pPIC9K-WT

[0104] The pPIC9K-WT and pPIC9K-118-160-3 pure enzyme solutions obtained under the optimized culture conditions were dialyzed into storage buffer (100 mM KPB, 0.2 mM MgCl2, 5% sucrose, 2.5% trehalose, pH 7.0) and placed in a 65°C incubator for accelerated stability testing. The residual enzyme activity was tested every 5 minutes. Figure 7 shown.

[0105] from Figure 7 It can be seen that the pPIC9K-118-160-3 mutant enzyme solution can still retain more than 60% of the enzyme activity after 50 minutes, and the enzyme activity decreases gently and slowly during constant temperature detection, while the enzyme activity of pPIC9K-WT has dropped to below 20% after 50 minutes, with a sharp decline and a fast rate; the ability of the pPIC9K-118-160-3 mutant to resist high temperatures is far superior to that of pPIC9K-WT.

[0106] 4. pH tolerance test of enzyme solution obtained from pPIC9K-118-160-3 mutant

[0107] The pure enzyme solutions of pPIC9K-WT and pPIC9K-118-160-3 obtained under the optimized culture conditions were dialyzed into 0.1M acetic acid solution at pH 4.0-6.0, 0.1M potassium phosphate solution at pH 6.0-8.0, 0.1M Tris-HCl at pH 7.5-9.0, and 0.1M carbonate solution at pH 8.5-10.5, respectively. The residual enzyme activity was measured at 20°C for 24 h. Figure 8 shown.

[0108] At the same pH (buffer), the enzyme activity obtained from the pPIC9K-WT strain was lower than that obtained from the pPIC9K-118-160-3 mutant strain, that is, the pH adaptation range of the mutant enzyme was wider than that of the pPIC9K-WT strain.

[0109] In summary, the pPIC9K-118-160-3 mutant strain and the enzyme solution obtained from the mutant strain have diverse enzymatic substrates and are not only used in blood glucose testing, but also break through the application limitations of blood glucose testing and provide good enzyme raw materials for the future use of one enzyme for multiple purposes and one enzyme for multiple tests. At the same time, it will also broaden the application of the enzyme in different industries and environments, improving it in depth and breadth.

[0110] 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-7, characterized in that: The amino acid sequence of the glucose dehydrogenase mutant pTrc99A-7 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-7 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-7 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-7 according to claim 1, characterized in that: Obtained by fermentation of the cell according to claim 6 or 7.

9. The preparation method according to claim 8, characterized in that: The fermentation conditions include: an initial inoculum amount of OD600=0.5-1.5, a pH of 6.5-7.5, a temperature of 25-30° C., and adding 1.5% methanol of the volume of the culture medium every 24 hours.

10. Use of the glucose dehydrogenase mutant pTrc99A-7 according to claim 1 in the catalysis of glucose, maltose, galactose, xylose, fructose or lactose.

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