Glucose dehydrogenase mutants with enhanced stability and activity

Abstract

The invention relates to the technical field of bioengineering, and discloses a glucose dehydrogenase mutant with enhanced stability and activity. The method comprises the following steps: firstly, carrying out molecular docking on glucose dehydrogenase and a glucose substrate to obtain a series of amino acid sites possibly influencing the enzyme structure and the combination of the enzyme and the substrate, then carrying out virtual saturated mutation on amino acids of the sites, and screening out mutation possibly enhancing the enzyme stability and activity through molecular dynamics simulation. A plurality of amino acid sites and mutation schemes capable of improving the stability and activity of the glucose dehydrogenase are obtained through screening after large-scale expression of the glucose dehydrogenase, the stability and activity of mutants obtained through single-point mutation and multi-point combined mutation of the sites are improved to different degrees, and the mutants can be used for preparing the glucose dehydrogenase mutant. The compound has high activity, can improve detection sensitivity and accuracy when used for detecting blood glucose, has high stability, is suitable for preparing kits and the like, and has wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a glucose dehydrogenase mutant with enhanced stability and activity. Background Art

[0002] With the continuous improvement of living conditions, the incidence of diabetes has continued to rise. Timely monitoring of blood sugar levels is the key to controlling the progression of diabetes. In the past, traditional blood sugar testing mostly used glucose oxidase (GOD), which has a significant dependence on dissolved oxygen in the test and has cross-reactions with other sugars, which may lead to inaccurate measurements. Glucose dehydrogenase (GDH) with flavin adenine dinucleotide (FAD) as a cofactor uses FAD as an electron acceptor, is independent of oxygen, and has low cross-reactivity to other sugars, making it show better accuracy and specificity in blood sugar testing. Therefore, glucose dehydrogenase is increasingly replacing glucose oxidase for blood sugar monitoring. At present, FAD-GDH used for blood sugar testing is mainly derived from Aspergillus, and efficient recombinant expression in yeast has been achieved, but there is still room for improvement in stability and activity. Modification to further improve stability and activity can further reduce production costs. Summary of the invention

[0003] In order to solve the above existing problems, the present invention discloses a glucose dehydrogenase mutant with enhanced stability and activity. Firstly, glucose dehydrogenase and a glucose substrate are molecularly docked to obtain a series of amino acid sites that may affect the enzyme structure and the binding of the enzyme to the substrate, and then the amino acids at these sites are virtually saturated mutated, and mutations that may enhance the enzyme stability and activity are screened out through molecular dynamics simulation. Then, wet experiments are started and screened after large-scale expression to obtain multiple amino acid sites and mutation schemes that improve the stability and activity of glucose dehydrogenase. The mutants obtained by single-point mutation and multi-point combination mutation at these sites have different degrees of improvement in stability and activity.

[0004] In one aspect, the present invention provides a glucose dehydrogenase mutant, wherein the glucose dehydrogenase mutant uses flavin adenine dinucleotide as a cofactor, and the glucose dehydrogenase mutant is as shown in Seq ID Amino acid mutations are performed based on the amino acid sequence shown in No.1, and the amino acid mutations include any one of the following mutations or a combination of mutations at any of the following mutation sites: G53N, G55R, Y98R, W117E, G163R, P303C, V310H, D316L, Q317R, F318R, N320I, G321R, S334K, S334M, S334F, S334W, T335I, Q377M, S413M, S413V, E414R, E414M, A474C, K475I, S503P, P507R, V551E, V551I, V551M, and V551C.

[0005] Protein sequence refers to the order of amino acid residues in a protein molecule, and amino acid mutation refers to the replacement of individual amino acids in a protein sequence. Amino acid mutation usually affects the local structure of a protein. Local structure refers to the local chemical structure, where new amino acids may form new hydrogen bonds with adjacent amino acids in space, and local spatial structure, such as affecting the spatial configuration of local peptide chains. Therefore, amino acid mutations at certain sites may change the properties and functions of a protein. Since amino acid mutations are local, it is understandable that benign amino acid mutations at multiple different sites can jointly enhance certain benign properties of the overall protein molecule by affecting different local structures.

[0006] The mutation site is represented by the amino acid code: amino acid before mutation + mutation site + amino acid after mutation; for example, A24G means that in a certain protein sequence, the mutation site is the 24th amino acid, which mutates from alanine (A) to glycine (G); the names and abbreviations of amino acids are publicly available and will not be described here.

[0007] Therefore, the meaning of "combination of mutations at any number of mutation sites" is: a combination of mutations occurring at different mutation sites. For example, S334K, S334M, S334F, and S334W are all mutations occurring at the 334th amino acid, which obviously cannot be considered as a combination. Another example is that G321R and S334K are mutations occurring at the 321st and 334th amino acids, respectively, which can be considered as a combination.

[0008] In some embodiments, the amino acid mutation comprises any one of the following mutations or a combination of mutations at any of the following mutation sites: G321R, S334K, S334M, S413M, S413V, S503P.

[0009] The present invention proves through experiments that mutations of G321R, S334K, S334M, S413M, S413V and S503P can effectively improve the activity of glucose dehydrogenase mutants, among which the activity of glucose dehydrogenase mutant with S413M mutation is the highest.

[0010] Preferably, the amino acid mutation includes S413M.

[0011] In some embodiments, the amino acid mutation comprises any one of the following mutations or a combination of mutations at any of the following mutation sites: G321R, S334K, S334M, S413V.

[0012] The present invention proves through experiments that the mutations of G321R, S334K, S334M and S413V can effectively improve the stability of the glucose dehydrogenase mutant and also can improve the activity. Among them, the glucose dehydrogenase mutant with the S413V mutation has the highest stability.

[0013] Preferably, the amino acid mutation comprises S413V.

[0014] On the other hand, the present invention provides a nucleic acid encoding the glucose dehydrogenase mutant.

[0015] In another aspect, the present invention provides a vector comprising the nucleic acid.

[0016] In another aspect, the present invention provides a cell, which is a host cell carrying the vector.

[0017] In some embodiments, the host cell is Pichia pastoris.

[0018] In another aspect, the present invention provides a method for preparing a glucose dehydrogenase mutant, comprising the steps of:

[0019] S1: synthesizing a FAD-GDH sequence optimized for codon usage frequency for Pichia pastoris, wherein the FAD-GDH sequence is the amino acid sequence shown in Seq ID No. 2, and inserting the FAD-GDH sequence into a vector;

[0020] S2: Using mutation primers, PCR amplification was performed with the vector obtained in S1 as the target to introduce the corresponding mutation, and the FAD-GDH mutant expression vector with correct sequencing was extracted and electro-transformed into the Pichia pastoris X-33 expression strain to screen and obtain the expression bacteria. The mutation primers include forward primers and reverse primers for any one of the following mutations or any multiple mutation sites:

[0021]

[0022]

[0023]

[0024] S3: fermentation, collection of fermentation supernatant, elution and purification.

[0025] On the other hand, the present invention provides a primer combination for preparing a glucose dehydrogenase mutant, comprising a forward primer and a reverse primer for any one of the following mutations or any of the following multiple mutation sites:

[0026]

[0027]

[0028] In still another aspect, the present invention provides a kit comprising the glucose dehydrogenase mutant.

[0029] In summary, the present invention includes at least one of the following beneficial technical effects:

[0030] 1. The present invention performs molecular docking on glucose dehydrogenase and glucose substrate to obtain a series of amino acid sites that may affect the enzyme structure and the binding of the enzyme to the substrate, and then performs virtual saturation mutation on the amino acids at these sites, and screens out mutations that may enhance the stability and activity of the enzyme through molecular dynamics simulation, and experimentally verifies that the mutations at the above amino acid sites have the effect of enhancing the stability and activity of the enzyme;

[0031] 2. Through experimental screening, we obtained preferred amino acid mutation sites with higher activity than the wild type. The mutants obtained by single-point mutation and multi-point combination mutation at these sites have higher activity and affinity, and can be used to detect blood sugar to improve the sensitivity and accuracy of detection;

[0032] 3. Through experimental screening, preferred amino acid mutation sites with higher stability than the wild type were obtained. The mutants obtained by single-point mutation and multi-point combination mutation at these sites have higher thermal stability. At the same time, according to activity experiments, they also have higher activity and affinity. They can be used to detect blood glucose to improve detection sensitivity and accuracy, and have higher stability, making them suitable for preparing test kits, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 :Molecular docking simulation diagram of FAD-GDH and D-glucose;

[0034] Figure 2 : Molecular dynamics simulation heat map of FAD-GDH binding affinity;

[0035] Figure 3 : FAD-GDH stability simulation heat map;

[0036] Figure 4 : Heat map of FAD-GDH stability and affinity simulation;

[0037] Figure 5 :Fermentation supernatant sampling and detection electrophoresis;

[0038] Figure 6 :Purification electrophoresis detection diagram;

[0039] Figure 7 :Results of FAD-GDH wild type and mutant activity assay;

[0040] Figure 8 : Results of measuring the activity changes of wild-type and mutant FAD-GDH after heat treatment at 50°C. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, which are only used to explain the present invention and are not intended to limit the scope of the present invention. The materials, reagents, etc. used in the following examples are reagents and materials available from commercial sources unless otherwise specified.

[0042] Example 1: Molecular docking, molecular dynamics simulation and virtual screening of FAD-GDH

[0043] The present invention uses the amino acid sequence of glucose dehydrogenase from Aspergillus flavus with FAD as a cofactor, the specific sequence is shown in Seq ID No. 1, and protein sequence analysis is performed based on this sequence.

[0044] Sequence 1 was compared in the PDB database, and a homologous crystal structure 4YNT with high sequence similarity was obtained. Based on this crystal structure, AlphaFold was used to obtain the complex structure of FAD cofactor and GDH by superposition. AutoDock Vina was further used to study the docking of FAD-GDH and D-glucose. According to the reported GDH active center, the docking box size was set to 25×25×25 cubic angstroms, the global search detail was 32, the regional parameters were kept as default, and the docking conformation with the highest score was output as the binding conformation. The binding mode of FAD-GDH and D-glucose obtained by molecular docking is shown in the figure. Figure 1 .

[0045] Among them, Gly-95, Tyr-54, Asn-319, His-506, Arg-502 and Asn-504 are the key amino acids in the active center. These amino acids interact with D-glucose through hydrogen bonds and are the main factors affecting the binding of the enzyme and the substrate. The sites near these amino acids and the amino acids at the key sites of the enzyme structure also affect the binding of the enzyme to the substrate and the stability of the enzyme structure itself. After analysis, the following possible amino acid sites are found: Gly-53, Gly-55, Tyr-98, Trp-117, Gly-163, Pro-303, Val-310, Asp-316, Gln-317, Phe-318, Asn-320, Gly-321, Ser-334, Thr-335, Gln-377, Ser-413, Glu-414, Ala-474, Lys-475, Ser-503, Pro-507, and Val-551.

[0046] Amber was used to simulate the molecular dynamics of the FAD-GDH and D-glucose complex. First, the charge of D-glucose was calculated, and then the force field was described. Hydrogen atoms and sodium ions and chloride ions were added to the system to balance the charge of the system. Then, energy optimization was performed, and isothermal isobaric ensemble simulation, isothermal isobaric equilibrium simulation, and isothermal isobaric ensemble simulation under boundary conditions were performed. The simulation was performed at 298.15K and 1atm, with an integration step of 2fs, and the trajectory was saved every 10ps for subsequent analysis.

[0047] The Residue Scanning Calculations module in the Schrodinger software package was used to perform amino acid scanning studies on the FAD-GDH and D-glucose complexes, calculate the output affinity change data, and display the analysis in the form of a heat map. Figure 2 Medium blue mutations indicate an improvement in FAD-GDH binding affinity, red indicates a decrease, and the depth of the color indicates the magnitude of the change. Figure 3 The medium blue epimutation has a positive effect on the stability of FAD-GDH, while red indicates a negative effect, and the depth of the color indicates the magnitude of the change. Figure 4 Blue indicates an improvement effect on FAD-GDH stability and no reduction effect on substrate affinity, and the depth of color indicates the magnitude of the change.

[0048] Taken together, the following mutations are present: G53N, G55R, Y98R, W117E, G163R, P303C, V310H, D316L, Q317R, F318R, N320I, G321R, S334K, S334M, S334F, S334W, T335I, Q377M, S413M, S413V, E414R, E414M, A474C, K475I, S503P, P507R, V551E, V551I, V551M, V551C.

[0049] Example 2: Site-directed mutagenesis and expression vector construction, transformation and expression, fermentation and purification of FAD-GDH

[0050] The codon usage frequency of the amino acid sequence Seq ID No.1 was optimized for Pichia pastoris to obtain the gene sequence Seq ID No.2, which was sent to a gene synthesis company for gene synthesis and inserted into the T cloning site of the pMD18-T cloning vector. The vector was named pMD18-T-GDH as the original vector for site-directed mutagenesis. The site-directed mutagenesis primers are shown in Table 1.

[0051] Table 1: Mutation Primers

[0052]

[0053]

[0054]

[0055] Using mutation primers, PCR amplification was performed with pMD18-T-GDH as the target to introduce the corresponding mutation. The reaction system is as follows:

[0056] 35 ng pMD18-T-GDH dsDNA

[0057] 5μl 10×pfu PCR buffer

[0058] 4 μl dNTP mix

[0059] 1.5 μl each of upstream and downstream primers

[0060] 1 μl pfu DNA polymerase

[0061] Make up to 50 μl with ddH2O.

[0062] PCR program: 95℃1min30s→(95℃30s→55℃1min→72℃8min)×18 cycles→16℃ insulation.

[0063] After the PCR reaction was completed and the temperature of the reaction system dropped below 37°C, 1 μl of Dpn I enzyme and 5.73 μl of 10× buffer were added and kept at 37°C for 1 hr.

[0064] After the reaction, take 5 μl of transformed DH5α competent cells, spread on ampicillin-resistant LB plates for overnight culture, and pick single clones from the plates for sequencing.

[0065] Using the correctly sequenced pMD18-T-GDH mutant vector as a template, the primers in Table 2 were used for PCR amplification to obtain the successfully mutated GDH gene fragment, and homologous recombination was performed with the pPICZαA vector treated with Xho I / Xba I double restriction enzymes, and Zeocin-resistant low-salt LB plates were coated for overnight culture, and single clones were picked from the plates for sequencing. The correctly sequenced FAD-GDH mutant expression vector was extracted using a midi plasmid extraction kit, linearized with Sac I endonuclease, and then electroporated into the Pichia pastoris X-33 expression strain to obtain the expression bacteria.

[0066] Table 2: Primers for GDH gene fragment

[0067] Primer name Primer sequences GDH-F 5'-aagaaggggtatctctcgagAAAAGAGCTAAGAATACAACGACATACG-3' GDH-R 5'-agatgagtttttgttctagaTTAAGCACTCTTGGCGTCTTCCTTA-3'

[0068] The wild-type and mutant Pichia pastoris expression strains of FAD-GDH were fermented in a 2.5L fermenter. The initial volume of BSM basal medium was 800ml, the inoculation volume of the fermentation starting seed solution was 40ml, and the culture was carried out at 30°C after inoculation. The dissolved oxygen electrode was calibrated to 100%, and the fermentation parameters were set after waiting for the parameters to stabilize: pH = 5.0 ± 0.1, ventilation ≥ 1vvm, dissolved oxygen ≥ 20%, and the stirring rate was set between 250rpm and 800rpm, and the dissolved oxygen was kept linked.

[0069] About 16 to 24 hours after inoculation, the glycerol in the basal culture medium was exhausted and the dissolved oxygen suddenly increased. Glycerol fed-batch culture was started with the feed rate set at 16 ml / hr and glycerol fed-batch culture for 4 hours.

[0070] After the glycerol feeding is completed, when the glycerol is exhausted and the dissolved oxygen rises sharply again, the culture temperature is lowered to 28°C and methanol induction culture is started. The flow acceleration of the methanol feed medium is 2.9 ml / min. After 2 hours of flow addition, the flow acceleration of the methanol feed medium is increased to 5.8 ml / min, and the flow addition is continued for 2 hours, and it is increased to 8.6 ml / min again. The flow addition is continued for 12 hours, and the increase to 9.5 ml / min is increased again to 10.5 ml / min after 12 hours of flow addition, and the increase to 11.5 ml / min is continued for 12 hours until the fermentation is completed. The supernatant of the fermentation is sampled and tested for electrophoresis diagrams after 20 hr, 44 hr, 68 hr and 92 hr of induction. Taking the wild type FAD-GDH as an example, the electrophoresis diagram is shown in Figure 5 .

[0071] After the fermentation is completed, the fermentation supernatant is collected by centrifugation, concentrated using a 10kD ultrafiltration membrane, and replaced with 1×PB pH7.0. The eluted target protein is purified using BioCap Q ion chromatography filler from Suzhou Bio-Gen Biotechnology Co., Ltd., and the solution is replaced with 50mM potassium phosphate buffer at pH6.5. Taking the wild type FAD-GDH as an example, the electrophoresis detection diagram after purification is as follows Figure 6 .

[0072] Example 3: Determination of FAD-GDH activity

[0073] The present invention adopts the DCIP method to determine the activity, and the activity is defined as: at 37° C. and pH 6.5, the amount of enzyme required to catalyze the reduction of 1 μmol DCIP per minute is defined as 1U.

[0074] A 1 mg / ml enzyme sample to be tested was diluted 1500 to 4000 times using 50 mM pH 6.5 phosphate buffer as a test sample.

[0075] Prepare reaction buffer (keep away from light, prepare on ice, and prepare immediately before use):

[0076] Reagent I: 50 mM PIPES-NaOH buffer pH 6.5 1025 μl

[0077] Reagent II: 1M D-glucose solution 295 μl

[0078] Reagent III: 24 mM phenazine methyl sulfate (PMS) 100 μl

[0079] Reagent IV: 2mM 2,6-dichloroindophenol sodium salt (DCIP) 50μl

[0080] Take 300μl of reaction buffer and add it to one well of a 96-well plate, and incubate at 37℃ for 5min. Add 10μl of the sample to be tested to each reaction well, mix well, incubate at 37℃, measure the absorbance at 600nm, and calculate the change rate of the absorbance value within 1min ΔAs; replace the sample to be tested with a blank control, and follow the same steps, measure the absorbance at 600nm at 37℃, and calculate the change rate of the absorbance value within 1min ΔAb. The activity of the enzyme sample to be tested can be calculated according to the following formula:

[0081]

[0082] Where:

[0083] S: GDH activity, unit is GDH activity unit per milliliter (U / mL)

[0084] ΔA: Change in absorbance after consumption of substrate, ΔA = ΔAs - ΔAb

[0085] df: dilution factor;

[0086] 16.8: The micromolar extinction coefficient of DCIP at 600 nm under the conditions of this experiment (cm 2 / μmol);

[0087] l: optical path length, usually 1 cm

[0088] The activities of different mutants are shown in Figure 7 (With the wild-type activity as 100%), the results showed that the activities of the mutants G321R, S334K, S334M, S413M, S413V and S503P were higher than the wild-type, among which the S413M mutant had the highest activity, which was 255% of the wild-type.

[0089] It can be seen that the mutations of G321R, S334K, S334M, S413M, S413V and S503P are beneficial to improving the activity. From a theoretical point of view, this is because the corresponding mutations change the local structure of the protein, thereby improving the docking of the mutant enzyme with the substrate D-glucose. From a theoretical point of view, it can be understood that the mutants obtained by combining the above mutations (except for multiple mutations at the same site) also have higher activity than the wild type.

[0090] Example 4: Determination of FAD-GDH stability

[0091] The wild type and each mutant were treated at 50°C for 15 min, 30 min and 60 min, and then the activity was measured according to the method of Example 3 to obtain the thermal stability of the wild type and each mutant at 50°C. The results are as follows: Figure 8 Although a series of mutants had higher activity than the wild type at 15 min, only the mutants of G321R, S334K, S334M and S413V had higher activity than the wild type throughout the whole process. The thermal stability (heat treatment activity) of 321R, S334K, S334M, S413V and the wild type was calculated based on the wild type activity as 100%. Table 3 shows.

[0092] Table 3: Thermal stability of G321R, S334K, S334M, S413V and wild type

[0093]

[0094]

[0095] It can be seen that the mutations of G321R, S334K, S334M and S413V are beneficial to improving thermal stability. From a theoretical point of view, this is because the corresponding mutations change the local structure of the protein. For example, new hydrogen bonds are introduced at the mutation site. The interaction of hydrogen bonds may fix the loop region, which contributes to the high thermal stability of the entire protein. From a theoretical point of view, it can be understood that the mutants obtained by combining the above mutations (except for multiple mutations at the same site) also have higher activity than the wild type.

[0096] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto, and any equivalent modifications or substitutions that can be easily thought of by any technician familiar with the technical field within the technical scope disclosed by the present invention should be included in the protection scope of the present invention.

[0097] Sequence Listing

[0098] Seq ID No.1

[0099]

[0100] Seq ID No.2

[0101]

Claims

1. A glucose dehydrogenase mutant, characterized in that: The glucose dehydrogenase mutant uses flavin adenine dinucleotide as a cofactor, and the glucose dehydrogenase mutant undergoes amino acid mutation based on the amino acid sequence shown in Seq ID No. 1, and the amino acid mutation includes any one of the following mutations or a combination of mutations at any of the following mutation sites: G53N, G55R, Y98R, W117E, G163R, P303C, V310H, D316L, Q317R, F318R, N320I, G321R, S334K, S334M, S334F, S334W, T335I, Q377M, S413M, S413V, E414R, E414M, A474C, K475I, S503P, P507R, V551E, V551I, V551M, and V551C.

2. The glucose dehydrogenase mutant according to claim 1, characterized in that The amino acid mutation includes any one of the following mutations or a combination of mutations at any of the following mutation sites: G321R, S334K, S334M, S413M, S413V, S503P.

3. The glucose dehydrogenase mutant according to claim 1, characterized in that The amino acid mutation includes any one of the following mutations or a combination of mutations at any of the following mutation sites: G321R, S334K, S334M, S413V.

4. A nucleic acid, characterized in that The nucleic acid encodes the glucose dehydrogenase mutant according to any one of claims 1 to 3.

5. A carrier, characterized in that The vector comprises the nucleic acid of claim 4.

6. A cell, characterized in that The cell is a host cell carrying the vector according to claim 5.

7. The cell according to claim 6, characterized in that The host cell is Pichia pastoris.

8. A method for preparing a glucose dehydrogenase mutant, characterized in that: Includes steps: S1: synthesizing a FAD-GDH sequence optimized for codon usage frequency for Pichia pastoris, wherein the FAD-GDH sequence is the amino acid sequence shown in Seq ID No. 2, and inserting the FAD-GDH sequence into a vector; S2: Using mutation primers, PCR amplification is performed with the vector obtained in S1 as the target to introduce the corresponding mutation, and the FAD-GDH mutant expression vector with correct sequencing is extracted and electroporated into the Pichia pastoris expression strain to obtain the expression bacteria, wherein the mutation primers include forward primers and reverse primers for any one of the following mutations or any multiple mutation sites: G53N-F 5'-agatccgaaagctaatccgtagttgttagcgttggtgacgtcagg-3' G53N-R 5'-cctgacgtcaccaacgctaacaactacggattagctttcggatct-3' G55R-F 5'-gcagatccgaaagctaaacggtaaccgttagcgttgg-3' G55R-R 5'-ccaacgctaacggttaccgtttagctttcggatctgc-3' Y98R-F 5'-gtacatcttcggctcgggtacgagccattccattaatcgtac-3' Y98R-R 5'-gtacgattaatggaatggctcgtacccgagccgaagatgtac-3' W117E-F 5'-ggtaacaggtccttccaagtctcaccttcgttaccaagtttttg-3' W117E-R 5'-caaaaacttggtaacgaaggtgagacttggaaggacctgttacc-3' G163R-F 5'-ccagaagctaatgagcgggaccagccaactttt-3' G163R-R 5'-aaaagttggctggtcccgctcattagcttctgg-3' P303C-F 5'-tttcgccaactgttggtaaatcaactctgcaggtaatattattttttttcaggatagtag-3' P303C-R 5'-ctactatcctgaaaaaaaataatattacctgcagagttgatttaccaacagttggcgaaa-3' V310H-F 5'-gcaagttttcgccatgtgttggtaaatcaactcttggggtaat-3' V310H-R 5'-attaccccaagagttgatttaccaacacatggcgaaaacttgc-3' D316L-F 5'-tccggccattccattattaaattgcaattgcaagttttcgccaactgttgg-3' D316L-R 5'-ccaacagttggcgaaaacttgcaattgcaatttaataatggaatggccgga-3' Q317R-F 5'-ctccggccattccattattaaaacgatcttgcaagttttcgccaac-3' Q317R-R 5'-gttggcgaaaacttgcaagatcgttttaataatggaatggccggag-3' F318R-F 5'-tctccggccattccattattacgttgatcttgcaagttttcgcc-3' F318R-R 5'-ggcgaaaacttgcaagatcaacgtaataatggaatggccggaga-3' N320I-F 5'-ctccggccattccaatattaaattgatcttgcaagttttcgc-3' N320I-R 5'-gcgaaaacttgcaagatcaatttaatattggaatggccggag-3' G321R-F 5'-gccctctccggccatacgattattaaattgatcttgcaagttttcg-3' G321R-R 5'-cgaaaacttgcaagatcaatttaataatcgtatggccggagaggc-3' S334K-F 5'-atactggggtatgtgactgtcttagctccagccaaaacaccatag-3' S334K-R 5'-ctatggtgttttggctggagctaagacagtcacataccccagtat-3' S334M-F 5'-atactggggtatgtgactgtcatagctccagccaaaacaccatag-3' S334M-R 5'-ctatggtgttttggctgagctatgacagtcacatacccagtat-3' S334F-F 5'-ctggggtatgtgactgtgaaagctccagccaaaacacca-3' S334F-R 5'-tggtgttttggctggagctttcacagtcacataccccag-3' S334W-F 5'-ctggggtatgtgactgtccaagctccagccaaaacacc-3' S334W-R 5'-ggtgttttggctggagcttgacagtcacataccccag-3' T335I-F 5'-atactggggtatgtgacgatagaagctccagccaaaacacc-3' T335I-R 5'-ggtgttttggctggagctctctatcgtcacataccccagtat-3' Q377M-F 5'-gtctttccaaatcctccattttcatgtgaccgttggaaaccta-3' Q377M-R 5'-tagtttccaacggtcacatgaaaatggaggattggaaagac-3' S413M-F 5'-tggcagtagtccccaaaattccatagatacagcatttcctcc-3' S413M-R 5'-GGGAGAATGCTGTACTATGAATTTGGGACTGCC-3' S413V-F 5'-gtccccaaaattcaacagatacagcatttcctcctccaggat-3' S413V-R 5'-atcctggaggaggaaatgctgtatctgttgaatttttgggac-3' E414R-F 5'-gaatggcagtagtccccaaaaacgagaagatacagcatttcctcc-3' E414R-R 5'-ggagaaatgctgtatctctcgtttttggggactactgccattc-3' E414M-F 5'-cgaatggcagtagtccccaaacatagaagatacagcatttcctcct-3' E414M-R 5'-aggagaaatgctgtatcttctatgttttggggactactgccattcg-3' A474C-F 5'-aggcttggtctctttgcaaatcagcttattcaagggggcagatct-3' A474C-R 5'-agactctgccccctgaataagctgatttgcaaagagaccagcct-3' K475I-F 5'-agtccaggctttggtctcaattgcaatcagcttattcaagggg-3' K475I-R 5'-ccccttgaataagctgattgcaattgagaccaagcctgact-3' S503P-F 5'-cagtgccaactgggtgaaagttcggtctgtagttagccttaaccac-3' S503P-R 5'-gtggttaaagctaactacagaccgaactttcacccagttggcactg-3' P507R-F 5'-catggcagcagtgccaacacggtgaaagttactctctgt-3' P507R-R 5′-acagaagtaactttcaccgtgttggcactgctgccatg-3′ V551E-F 5'-cggcgtacaatgtggactctagatgtccacaaacc-3' V551E-R 5'-ggtttgtggacatcttagtccacattgtacgcg-3' V551I-F 5'-cggcgtacaatgtggagattagatgtccacaaacctgaaatg-3' V551I-R 5'-catttcaggtttgtggacatctaatctccacattgtacgccg-3' V551M-F 5'-gcgtacaatgtggcaattagatgtccacaaacctgaa-3' V551M-R 5'-ttcaggtttgtggacatctaatgtccacattgtacgc-3' V551C-F 5'-cacggcgtacaatgtggagcatagatgtccacaaacctgaaatggt-3' V551C-R 5'-accatttcaggtttgtggacatctatgctccacattgtacgccgtg-3'; S3: fermentation, collection of fermentation supernatant, elution and purification.

9. A primer combination for preparing a glucose dehydrogenase mutant, characterized in that: Forward primers and reverse primers for any of the following mutations or any of the following mutation sites: G53N-F 5'-agatccgaaagctaatccgtagttgttagcgttggtgacgtcagg-3' G53N-R 5'-cctgacgtcaccaacgctaacaactacggattagctttcggatct-3' G55R-F 5'-gcagatccgaaagctaaacggtaaccgttagcgttgg-3' G55R-R 5'-ccaacgctaacggttaccgtttagctttcggatctgc-3' Y98R-F 5'-gtacatcttcggctcgggtacgagccattccattaatcgtac-3' Y98R-R 5'-gtacgattaatggaatggctcgtacccgagccgaagatgtac-3' W117E-F 5'-ggtaacaggtccttccaagtctcaccttcgttaccaagtttttg-3' W117E-R 5'-caaaaacttggtaacgaaggtgagacttggaaggacctgttacc-3' G163R-F 5'-ccagaagctaatgagcgggaccagccaactttt-3' G163R-R 5'-aaaagttggctggtcccgctcattagcttctgg-3' P303C-F 5'-tttcgccaactgttggtaaatcaactctgcaggtaatattattttttttcaggatagtag-3' P303C-R 5'-ctactatcctgaaaaaaaataatattacctgcagagttgatttaccaacagttggcgaaa-3' V310H-F 5'-gcaagttttcgccatgtgttggtaaatcaactcttggggtaat-3' V310H-R 5'-attaccccaagagttgatttaccaacacatggcgaaaacttgc-3' D316L-F 5'-tccggccattccattattaaattgcaattgcaagttttcgccaactgttgg-3' D316L-R 5'-ccaacagttggcgaaaacttgcaattgcaatttaataatggaatggccgga-3' Q317R-F 5'-ctccggccattccattattaaaacgatcttgcaagttttcgccaac-3' Q317R-R 5'-gttggcgaaaacttgcaagatcgttttaataatggaatggccggag-3' F318R-F 5'-tctccggccattccattattacgttgatcttgcaagttttcgcc-3' F318R-R 5'-ggcgaaaacttgcaagatcaacgtaataatggaatggccggaga-3' N320I-F 5'-ctccggccattccaatattaaattgatcttgcaagttttcgc-3' N320I-R 5'-gcgaaaacttgcaagatcaatttaatattggaatggccggag-3' G321R-F 5'-gccctctccggccatacgattattaaattgatcttgcaagttttcg-3' G321R-R 5'-cgaaaacttgcaagatcaatttaataatcgtatggccggagagggc-3' S334K-F 5'-atactggggtatgtgactgtcttagctccagccaaaacaccatag-3' S334K-R 5'-ctatggtgttttggctggagctaagacagtcacataccccagtat-3' S334M-F 5'-atactggggtatgtgactgtcatagctccagccaaaacaccatag-3' S334M-R 5'-ctatggtgttttggctgagctatgacagtcacatacccagtat-3' S334F-F 5'-ctggggtatgtgactgtgaaagctccagccaaaacacca-3' S334F-R 5'-tggtgttttggctggagctttcacagtcacataccccag-3' S334W-F 5'-ctggggtatgtgactgtccaagctccagccaaaacacc-3' S334W-R 5'-ggtgttttggctggagcttgacagtcacataccccag-3' T335I-F 5'-atactggggtatgtgacgatagaagctccagccaaaacacc-3' T335I-R 5'-ggtgttttggctggagctctctatcgtcacataccccagtat-3' Q377M-F 5'-gtctttccaaatcctccattttcatgtgaccgttggaaaccta-3' Q377M-R 5'-tagtttccaacggtcacatgaaaatggaggattggaaagac-3' S413M-F 5'-tggcagtagtccccaaaattccatagatacagcatttcctcc-3' S413M-R 5'-GGGAGAATGCTGTACTATGAATTTGGGACTGCC-3' S413V-F 5'-gtccccaaaattcaacagatacagcatttcctcctccaggat-3' S413V-R 5'-atcctggaggaggaaatgctgtatctgttgaatttttgggac-3' E414R-F 5'-gaatggcagtagtccccaaaaacgagaagatacagcatttcctcc-3' E414R-R 5'-ggagaaatgctgtatctctcgtttttggggactactgccattc-3' E414M-F 5'-cgaatggcagtagtccccaaacatagaagatacagcatttcctcct-3' E414M-R 5'-aggagaaatgctgtatcttctatgttttggggactactgccattcg-3' A474C-F 5'-aggcttggtctctttgcaaatcagcttattcaagggggcagatct-3' A474C-R 5'-agatctgcccccttgaataagctgatttgcaaagagaccaagcct-3' K475I-F 5'-agtccaggcttggtctcaattgcaatcagcttattcaagggg-3' K475I-R 5'-ccccttgaataagctgattgcaattgagaccaagcctggact-3' S503P-F 5'-cagtgccaactgggtgaaagttcggtctgtagttagcctttaaccac-3' S503P-R 5'-gtggttaaaggctaactacagaccgaactttcacccagttggcactg-3' P507R-F 5'-catggcagcagtgccaacacggtgaaagttacttctgt-3' P507R-R 5'-acagaagtaactttcaccgtgttggcactgctgccatg-3' V551E-F 5'-cggcgtacaatgtggactctagatgtccacaaacc-3' V551E-R 5'-ggtttgtggacatctagagtccacattgtacgccg-3' V551I-F 5'-cggcgtacaatgtggagattagatgtccacaaacctgaaatg-3' V551I-R 5'-catttcaggtttgtggacatctaatctccacattgtacgccg-3' V551M-F 5'-gcgtacaatgtggacattagatgtccacaaacctgaa-3' V551M-R 5'-ttcaggtttgtggacatctaatgtccacattgtacgc-3' V551C-F 5'-cacggcgtacaatgtggagcatagatgtccacaaacctgaaatggt-3' V551C-R 5'-accatttcaggtttgtggacatctatgctccacattgtacgccgtg-3.

10. A kit, characterized in that: Comprising the glucose dehydrogenase mutant according to any one of claims 1-3.

Images