Glucose dehydrogenase gox-2015 mutant and application thereof

Abstract

This invention discloses a glucose dehydrogenase GOX-2015 mutant and its applications, relating to the field of bioengineering technology. This invention designs and modifies the amino acid sequence of wild-type glucose dehydrogenase GOX-2015, providing 18 single mutation sites and 16 combined mutation sites. The resulting GOX-2015 mutant exhibits approximately 2-13°C higher thermostability and approximately 1.2-4.5 times higher activity compared to wild-type GOX-2015. Considering the significant improvements in thermostability and activity from the mutation sites, this invention also provides a preferred mutant protein, which, compared to wild-type GOX-2015, exhibits higher yield, activity, and thermostability, has broader application prospects, and is more suitable for the efficient production of NADPH coenzyme via biotransformation, thus facilitating large-scale production and industrial applications.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a glucose dehydrogenase GOX-2015 mutant and its applications. Background Technology

[0002] Glucose dehydrogenase (GDH) is an important oxidoreductase widely used in biosensors, medical diagnostics, the food industry, and biofuels. It catalyzes the oxidation of glucose to gluconolactone, accompanied by the reduction of coenzymes (such as NAD+ or PQQ). This process is particularly important in biosensing, especially in blood glucose monitoring systems, where GDH serves as a key enzyme for the quantitative detection of glucose.

[0003] In recent years, with the rapid development of biotechnology, the requirements for the thermal stability and catalytic activity of enzymes have been increasing. Enzymes with high thermal stability can maintain their activity at high temperatures, making them suitable for high-temperature reaction conditions in industrial production, while also extending their lifespan and reducing production costs. Furthermore, high catalytic activity can improve reaction efficiency, reduce enzyme dosage, further lower costs, and enhance detection sensitivity.

[0004] Although various glucose dehydrogenases have been developed and applied in actual production, they still have certain limitations in terms of thermal stability and catalytic activity. For example, while existing GDH mutants exhibit good performance under specific conditions, their activity tends to decrease under high temperature or long-term reaction conditions, limiting their application in a wider range of fields.

[0005] Currently, although many glucose dehydrogenases have been reported, most exhibit poor thermostability and activity. GOX-2015, a glucose dehydrogenase derived from *Staphylococcus aureus*, has attracted significant attention due to its higher NADP+-dependent coenzyme activity and better thermostability compared to other glucose dehydrogenases. Previous research (CN114836396A) disclosed a glucose dehydrogenase mutant, its protein crystal, and its applications. This mutant, D259C, mutates the aspartic acid D at position 259 of the wild-type glucose dehydrogenase protein sequence to cysteine ​​C. Compared to the wild-type protein, the D259C mutant exhibits higher thermostability (Tm value: 64℃ for wild-type, 73℃ for mutant). The yield of the D259C mutant is nearly twice that of the wild-type protein (67.2 mg / L), achieving a dual improvement in thermostability and yield. Furthermore, the NADP+ reduction activity of the D259C mutant is 3.5 times higher than other reported glucose dehydrogenases; however, the previous study did not compare the activities of the wild-type protein and the D259C mutant. To further expand the application areas of glucose dehydrogenase, developing glucose dehydrogenase mutants with higher thermal stability, catalytic activity, and high yield is of great practical significance. Summary of the Invention

[0006] The purpose of this invention is to enable glucose dehydrogenase to meet a wider range of application needs, especially for applications in high-temperature industrial environments and long-term reaction conditions, and to provide a glucose dehydrogenase GOX-2015 mutant and its applications.

[0007] The present invention achieves the above objectives through the following technical solutions: As a first aspect of the present invention, a glucose dehydrogenase GOX-2015 mutant is provided, said glucose dehydrogenase GOX-2015 mutant being obtained by single-point mutation or combination mutation of the sequence of wild-type GOX-2015 as shown in SEQ ID NO.1; wherein, The single mutation site is any one of E62I, K77S, G115A, G115K, G115R, H158G, E137S, M201K, M201S, M201A, D205N, S225P, D230A, M240A, N263P, N263D, S265L, and S265F; The combined mutation sites are any one of the following (1)-(16): (1) M201K, H158G; (2) M201K, G115R; (3) M201K, S225P; (4) M201K, G115K; (5) M201K, G115A; (6) M201K, D230A; (7) M2 01K, G115K, H158G; (8) M201K, G115K, H158G, D259C; (9) M201K, G115K, H158G, D205N; (10) M201K, G115K, H158G, K77 S; (11) M201K, G115K, H158G, E62I; (12) M201K, G115K, H158G, E137S; (13) M201K, G115K, H158G, D259C, D205N; (14) M201K, G115K, H158G, D259C, K77S; (15) M201K, G115K, H158G, D259C, E62I; (16) M201K, G115K, H158G, D259C, E137S.

[0008] A further improvement is that the glucose dehydrogenase GOX-2015 mutant is obtained by single-point mutation or combination mutation of the wild-type GOX-2015 sequence as shown in SEQ ID NO.1: The single mutation site is any one of M201K, E62I, K77S, E137S, and D205N; The combined mutation sites are any one of the following (1)-(5): (1) M201K, H158G; (2) M201K, G115R; (3) M201K, G115K; (4) M201K, G115K, H158G, E137S; (5) M201K, G115K, H158G, E62I.

[0009] A further improvement is that the combined mutation sites of the glucose dehydrogenase GOX-2015 mutant are M201K, G115K, H158G and E137S, and the amino acid sequence of the GOX-2015 mutant is shown in SEQ ID NO.3.

[0010] As a second aspect of the present invention, a method for preparing the glucose dehydrogenase GOX-2015 mutant as described above is also provided, comprising the following steps: (1) Based on the amino acid sequence of the glucose dehydrogenase GOX-2015 mutant, a gene sequence that can encode its amino acid sequence was designed and synthesized, and then the gene sequence was constructed on an expression vector to obtain a recombinant plasmid. (2) The recombinant plasmid obtained in step (1) is expressed using the Escherichia coli prokaryotic expression system to obtain the expression product. The glucose dehydrogenase GOX-2015 mutant is obtained by purifying the expression product.

[0011] As a third aspect of the invention, a polynucleotide is also provided, said polynucleotide encoding a glucose dehydrogenase GOX-2015 mutant as described in any of the above descriptions.

[0012] A further improvement is that the polynucleotide sequence encoding the amino acid sequence of the glucose dehydrogenase GOX-2015 mutant as shown in SEQ ID NO.3 is shown in SEQ ID NO.4.

[0013] As a fourth aspect of the present invention, a recombinant plasmid is provided, said recombinant plasmid being an expression vector capable of correspondingly translating and expressing the glucose dehydrogenase GOX-2015 mutant as described above.

[0014] A further improvement is that the expression vector is pET-28a.

[0015] As a fifth aspect of the present invention, a glucose dehydrogenase GOX-2015 mutant expression system is provided, which is Escherichia coli BL21(DE3) transformed with any of the recombinant plasmids described above.

[0016] As a sixth aspect of the present invention, an application is provided of the glucose dehydrogenase GOX-2015 mutant as described above as a catalytic enzyme in the production of NADPH coenzyme by biotransformation.

[0017] As a seventh aspect of the present invention, an application is provided of the glucose dehydrogenase GOX-2015 mutant as described above in enhancing the activity or thermostability of glucose dehydrogenase.

[0018] The present invention has the following beneficial effects: This invention designs and modifies the amino acid sequence of wild-type glucose dehydrogenase GOX-2015, providing a series of single-point mutation sites and combined mutation sites. The resulting GOX-2015 mutant exhibits approximately 2-13°C higher thermostability and approximately 1.2-4.5 times higher activity compared to wild-type GOX-2015. Considering the significant improvements in thermostability and activity from the mutation sites, this invention also provides a preferred GOX-2015 mutant protein (M201K, G115K, H158G, E137S), which, compared to wild-type GOX-2015, exhibits higher yield, activity, and thermostability, has broader application conditions, and is more suitable for the efficient production of NADPH coenzyme via biotransformation, thus facilitating large-scale production and industrial applications. Attached Figure Description

[0019] Figure 1 The modified pET-28a vector spectrum provided by this invention; Figure 2 The results of small-scale purification of the GOX-2015 single mutant protein provided by this invention in a cell-free expression system; Figure 3 The results of small-scale purification of the GOX-2015 combinatorial mutant protein provided by this invention in a cell-free expression system; Figure 4 The activity detection results of the GOX-2015 single mutant protein provided by this invention; Figure 5 The activity detection results of the GOX-2015 combined mutant protein provided by this invention; Figure 6 The affinity chromatography purification results of the GOX-2015 (M201K, G115K, H158G, E137S) mutant protein provided by this invention; Figure 7 The quality test results of the GOX-2015 (M201K, G115K, H158G, E137S) mutant protein provided by this invention; Figure 8 The activity detection results of wild-type GOX-2015 protein, GOX-2015 (M201K, G115K, H158G, E137S) mutant protein and GOX-2015 (D259C) mutant protein provided by the present invention. Detailed Implementation

[0020] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0021] 1. Materials and Reagents Unless otherwise specified, all reagents or instruments used in this invention are commercially available products.

[0022] 2. Method Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art. Where specific conditions are not specified, they shall be performed under conventional conditions or conditions recommended by the manufacturer.

[0023] 2.1 Construction of the GOX-2015 mutant plasmid This invention modifies the amino acid sequence of wild-type glucose dehydrogenase GOX-2015, whose Uniprot number is Q5FPE5, and its protein sequence is shown in SEQ ID NO.1. The gene sequences of wild-type GOX-2015 and its mutants were obtained through gene synthesis methods.

[0024] All mutants were constructed using molecular cloning methods with corresponding mutant primers designed based on the wild-type sequence. A total of 18 single-point mutations and 16 combined mutations were identified. The specific mutation site information is as follows: Single-point mutations include: E62I, K77S, G115A, G115K, G115R, E137S, H158G, M201K, M201S, M201A, D205N, S225P, D230A, S240A, N263P, N263D, S265L, and S265F.

[0025] Combinatorial mutations include: (M201K, H158G), (M201K, G115R), (M201K, S225P), (M201K, G115K), (M201K, G115A), (M201K, D230A), (M201K, G115K, H158G), (M201K, G115K, H158G, D259C), (M201K, G115K, H158G, D205N), (M201K, G115K, H158G, K 77S), (M201K, G115K, H158G, E62I), (M201K, G115K, H158G, E137S), (M201K, G115K, H158G, D259C, D205N), (M201K, G115K, H158G, D259C, K77S), (M201K, G115K, H158G, D259C, E62I) and (M201K, G115K, H158G, D259C, E137S).

[0026] Wild-type GOX-2015 and its mutant proteins were constructed using the modified pET-28 vector (GenScript). This vector incorporates an 8His-strepII-TEV-GG tag sequence fused to the T7 promoter. The tag sequence is shown in SEQ ID NO.2 (where 8His and strep II are tag sequences used for affinity purification, "TEV" is the TEV protease cleavage site used for tag removal during subsequent purification, and "GG" is the tag sequence itself). The gene sequences of the recombinant proteins were verified to be correct by the sequencing company. The vector map is shown below. Figure 1 .

[0027] 2.2 Expression and purification of GOX-2015 mutant protein 2.2.1 Low-level expression of GOX-2015 mutant protein The low-level expression of the GOX-2015 mutant protein was performed using a cell-free expression method. The cell-free expression protocol is described in [Levine, MZ, et al. (2019). Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology]. It mainly includes the following two steps: (1) Preparation of crude extract of Escherichia coli: BL21(DE3) bacterial culture was inoculated into 2×YT medium and cultured at 37℃ until OD 600 When the concentration reaches 0.6-0.8, add isopropyl β-D-1-thiogalactopyranoside (IPTG). OD 600 At approximately 3 minutes, centrifuge to collect the bacteria. Wash each gram of wet bacterial cells three times with S30 buffer (10 mM Tris-acetate, pH 8.2, 14 mM magnesium acetate, 50 mM potassium acetate, 2 mM DTT) at 4°C. Resuspend the bacterial cells in 1 mL of S30 buffer at a ratio of 1 g of bacterial cells. After sonicating the cells with an ultrasonic disruptor, centrifuge at 13000 × g for 10 minutes. Transfer the supernatant to a nuclease-free tube, flash freeze in liquid nitrogen, and store at -80°C.

[0028] (2) Expression of protein in cell-free system: Plasmid, cell extract, reaction buffer (phosphoenolpyruvate, PEG mixture, potassium glutamate, magnesium glutamate, amino glutamate, 20 amino acids, reaction energy substances (nicotinamide adenine dinucleotide, adenosine 5'-triphosphate disodium salt, cytidine disodium salt, guanosine 5'-monophosphate disodium hydrate and uridine disodium salt, leucovorin calcium salt and tRNA), glucose, spermidine and 1,4-diaminobutane, etc., were added to a 15 mL nuclease-free centrifuge tube according to the proportions in the literature. The reaction system was 200 μL, and the reaction conditions were 37℃, 200 rpm. After 4 hours, the reaction solution was centrifuged at 12000 rpm for 10 minutes, the supernatant and precipitate were collected, and the samples of each step were fixed with loading.

[0029] 2.2.2 Small-scale purification of GOX-2015 mutant protein (1) Small-scale purification of GOX-2015 single mutant protein After expression in the cell-free system, 50 μL of Strep-Tactin®XT packing material, which had been treated with buffer (25 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM TCEP), was added to the supernatant. The mixture was incubated at 4 °C for 30 minutes. The incubated sample was then centrifuged at 12,000 rpm at 4 °C for 10 minutes. 1 mL of buffer was added, and the sample was washed 3 times. Then, 100 μL of elution buffer (25 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM TCEP, 75 mM biotin) was added, and the sample was centrifuged at 12,000 rpm at 4 °C for 5 minutes. The eluted sample was collected. A small amount of the sample from each step was fixed with loading buffer and then analyzed by SDS-PAGE.

[0030] The experimental results are shown in Figure 2 Of the 18 single-point mutations, all single-point mutant proteins were clearly expressed, and their aggregation state was consistent with that of wild-type proteins as tetramer proteins.

[0031] (2) Small-scale purification of GOX-2015 combined mutant protein The purification method for combined mutants was the same as that for single-point mutants. Small-scale purification results showed that all 16 combined mutant proteins were clearly expressed in a cell-free system. FSEC results of the expressed samples showed that all combined mutant proteins aggregated in a tetramer state. Further purification was performed on all expressed samples.

[0032] The purification results are shown below. Figure 3 All samples can be eluted and are of high purity.

[0033] 2.3. Detection of thermal stability of GOX-2015 mutant protein The thermal stability test of the GOX-2015 mutant protein used protein thermal shift (ThermoFluor) technology. Utilizing the protein's structural characteristics, the protein contains hydrophobic regions hidden internally. As the temperature rises, the protein structure opens up, exposing these hydrophobic regions. The fluorescent dye SYPRO Orange can then bind to these regions, exciting fluorescence. A melting curve is formed based on the change in fluorescence signal intensity. The temperature corresponding to the maximum value of the derivative of the melting curve is the melting point (Tm). The more stable the protein, the higher the measured Tm value.

[0034] The specific steps are as follows: Add 5 μg of GOX-2015 mutant protein to each well of a 96-well PCR plate, and then add 10×SYPRO Orange fluorescent dye to the corresponding well. Place the 96-well PCR plate in a qPCR instrument, set the instrument parameters, and increase the temperature from 25℃ to 99℃ at a gradient of 1℃ per minute. Calculate the protein melting curve. The specific Tm values ​​for all single mutant proteins are shown in Table 1.

[0035] Table 1. Tm values ​​of GOX-2015 single mutant protein ; As shown in Table 1, among the 18 single mutant proteins, only the E62I and K77S mutations showed a relatively small increase in Tm values, while the others all showed an increase of at least 2°C. Specifically, the Tm values ​​of G115A, G115K, G115R, H158G, D230A, S225P, S265L, N263P, N263D, and S265F increased by 3-5°C. Therefore, this invention screened out 16 mutants—G115A, G115K, G115R, H158G, E137S, M201K, M201S, M201A, D205N, S225P, D230A, M240A, N263P, N263D, S265L, and S265F—that can improve the thermal stability of GOX-2015. Furthermore, the Tm values ​​show that amino acids G115, M201, N263, and S265 play an important role in protein thermal stability.

[0036] Single mutation sites with significant increases in M201K, G115K, G115A, G115R, H158G, D230A, and S225P were selected and combined. After small-scale expression and purification, their Tm values ​​were detected, and the results are shown in Table 2.

[0037] Table 2. Tm values ​​of GOX-2015 combined mutant proteins ; As shown in Table 2, the Tm values ​​of the above combined mutations were all increased by more than 5℃. Among them, the Tm value of (M201K, G115K, H158G) reached 74.7℃, which is 1.7℃ higher than the D259C (Tm=73℃) single mutant mentioned in the previous study. Therefore, the (M201K, G115K, H158G) mutation was combined with the D259C single mutant to obtain the (M201K, G115K, H158G, D259C) combined mutant. The Tm value of the protein of this combined mutant was 76.3℃, which was 3.3℃ higher than the Tm value of the D259C single mutant.

[0038] Furthermore, the Tm values ​​of the six combined mutations (M201K, G115K, H158G, E137S), (M201K, G115K, H158G, D259C, D205N), (M201K, G115K, H158G, D259C, K77S), (M201K, G115K, H158G, D205N), (M201K, G115K, H158G, D259C, E62I), and (M201K, G115K, H158G, D259C, E137S) are all higher than the Tm value of the D259C single mutant, indicating that the present invention provides a GOX-2015 mutant protein with higher thermal stability.

[0039] 2.4 Detection of GOX-2015 mutant protein activity To further verify the activity of the mutant proteins with improved thermostability, activity assays were performed on these mutant proteins. GOX-2015 is a glucose dehydrogenase that can convert NADP+ to NADPH using glucose as a substrate. The activity assay system provided in this invention uses glucose as a substrate, adds the coenzyme NADP+, and determines the enzyme activity of different GOX-2015 mutant proteins by detecting the fluorescence absorption signal of the generated NADPH at 340 nm.

[0040] The specific experimental procedures are as follows: (1) Preparation of buffer: 50 mM Tris pH 8.0, substrate 5 mM glucose, coenzyme 2 mM NADP+, reaction temperature 20 ℃. 30 μL of substrate and coenzyme NADP+ were transferred to a 384-well plate with two replicates. 50 nM of the GOX-2015 mutant protein to be tested was transferred to the corresponding well. The plate was immediately centrifuged and vortexed to mix. The fluorescence signal values ​​generated by the reaction were collected using a TECAN F200 microplate reader. Data analysis was performed using GraphPad Prism9 software to obtain the enzyme activity parameters of the tested protease.

[0041] (2) Results Analysis: According to the enzyme activity detection results, the activities of E62I, K77S, E137S, and D205N in the single mutant proteins increased by 3.2-4.5 times, the activity of M201K increased by 1.2 times, N263P, N263D, S265L, and S265F had almost no activity, and the activities of other mutant proteins remained between 50% and 75% of the wild-type protein activity. Figure 4 ).

[0042] The combinatorial mutant proteins include (M201K, G115K, H158G, D259C), (M201K, G115K, H158G, D259C, D205N), (M201K, G115K, H158G, D259C, E137S), (M201K, G115K, H158G, D259C, K77S), and (M201K, G115K, H158G, D259C, E62I). Five mutant proteins maintained only 10%-30% of the activity compared to the wild-type protein, while the other mutant proteins maintained 40% or more of the activity. Among them, (M201K, H158G), (M201K, G115R), (M201K, G115K), (M201K, G115K, H158G, E137S), and (M201K, G115K, H158G, E62I) had 1.2-1.7 times higher activity than the wild-type. Figure 5 These data indicate that D259C has a significant impact on the activity of the GOX-2015 protein; the activity of mutants without the D259C mutation is higher than that of mutants fused with D259C.

[0043] Based on the data on thermostability and activity, it can be seen that among the single mutations, M201K, E62I, K77S, E137S, and D205N are high-quality mutation sites that improve both thermostability and activity; among the combined mutations, (M201K, H158G), (M201K, G115R), (M201K, G115K), (M201K, G115K, H158G, E137S), and (M201K, G115K, H158G, E62I) are mutant proteins that improve both thermostability and activity.

[0044] 2.5 Expression and purification of GOX-2015 (M201K, G115K, H158G, E137S) mutant proteins To further investigate the function of the high-quality mutant protein GOX-2015, mutants of GOX-2015 (M201K, G115K, H158G, E137S) with significantly improved Tm values ​​and activities were selected for heterologous expression in Escherichia coli. The expressed protein was then purified, and its amino acid sequence is shown in SEQ ID NO.3 and its nucleotide sequence is shown in SEQ ID NO.4.

[0045] 2.5.1 Expression of GOX-2015 (M201K, G115K, H158G, E137S) mutant proteins After transforming the recombinant plasmid containing the GOX-2015 (M201K, G115K, H158G, E137S) protein into BL21(DE3) bacteria, the strain was inoculated into 50 ml of LB liquid medium and cultured overnight at 37°C. The overnight cultured bacteria were then inoculated into 1 L of LB liquid medium at a ratio of 1:100 and cultured at 37°C until the bacterial culture reached OD. 600 When the concentration is 0.6-0.8, add 0.5 mM IPTG, incubate overnight at 15°C, and collect the bacterial cells by centrifugation at 5000 rpm for purification.

[0046] 2.5.2 Purification of GOX-2015 (M201K, G115K, H158G, E137S) mutant proteins (1) Affinity chromatography The collected bacterial blocks were weighed and added to the appropriate volume of lysis buffer (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5% glycerol) at a 1:10 ratio. The bacterial cells were homogenized using a high-pressure homogenizer, and the supernatant was collected by centrifugation at 16,000 rpm. Proteins were enriched and purified using a Ni Bestarose FF affinity chromatography column. The specific procedure was as follows: the Ni Bestarose FF affinity chromatography column was first washed and equilibrated with lysis buffer to 10 column volumes. Then, the lysis supernatant was loaded onto the Ni Bestarose FF affinity chromatography column, and eluted with lysis buffer containing 50 mM imidazole. The eluted protein was collected for SDS-PAGE analysis, and the protein concentration was determined using Nanodrop to calculate the protein yield. The protein purification results are shown below. Figure 6 The yield was 100 mg / L. Previous studies have disclosed that the yield of wild-type protein is about 67.2 mg / L. It can be seen that the yield of GOX-2015 (M201K, G115K, H158G, E137S) mutant protein is nearly 1.5 times higher than that of wild type.

[0047] (2) Enzyme digestion and reverse affinity chromatography To obtain a protein with higher purity, a certain amount of TEV enzyme (Biortus, BP11748) was added to the sample after affinity chromatography. After overnight digestion at 4°C, the supernatant was further purified using a HisFF chromatography column. Since GOX-2015 (M201K, G115K, H158G, E137S) does not have an affinity tag after digestion, it will not bind to the affinity column and will flow out of the column (denoted as the permeate). Therefore, the permeate was collected.

[0048] (3) Gel filtration chromatography and QC detection The permeate was concentrated to approximately 2 mL and then subjected to gel filtration chromatography. The gel chromatography column was a Superdex 200 Increase 10 / 300GL, and the buffer was 25 mM HEPES (pH 7.5) and 100 mM NaCl. The gel filtration samples were collected and subjected to protein content determination, specifically SDS-PAGE purity analysis, mass spectrometry analysis, and analytical molecular sieve analysis. The results are shown below. Figure 7 As shown.

[0049] SDS-PAGE results showed that the purity of both the wild-type and GOX-2015 mutant proteins was greater than 99%. Mass spectrometry results also showed that the molecular weight of the tested samples was basically consistent with that of the target protein, indicating that the purified protein was the target protein. In addition, analytical molecular sieve results showed that all proteins were in a state close to tetramer in solution.

[0050] 2.5.3. Determination of the activity of GOX-2015 (M201K, G115K, H158G, E137S) mutants After obtaining high-purity GOX-2015 (M201K, G115K, H158G, E137S) mutants, their activities were compared with wild-type GOX-2015 (Biortus, BP15508-00A) and the D259C mutant protein (Biortus, BP17903-00A) from previous studies. The activity detection method was consistent with that in previous studies.

[0051] The activity test results are shown below. Figure 8 The (M201K, G115K, H158G, E137S) mutant protein exhibits 1.15 times higher activity than the wild type and 7 times higher activity than the D259C mutant protein, indicating that the (M201K, G115K, H158G, E137S) mutant protein provided by this invention has higher thermal stability and activity.

[0052] 3. Conclusion The GOX-2015 mutant protein provided by this invention has higher protein yield, higher enzyme activity and better thermal stability, has broader application conditions and stronger practical application value, and is more suitable for large-scale production and industrial use.

[0053] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A glucose dehydrogenase GOX-2015 mutant, characterized in that, The glucose dehydrogenase GOX-2015 mutant was obtained by single-point mutation or combination mutation of the wild-type GOX-2015 sequence as shown in SEQ ID NO.1; wherein, The single-point mutation site is E62I; The sites of the combined mutations are M201K, G115K, H158G, and E62I.

2. A method for preparing the glucose dehydrogenase GOX-2015 mutant as described in claim 1, characterized in that, Includes the following steps: (1) Based on the amino acid sequence of the glucose dehydrogenase GOX-2015 mutant, a gene sequence that can encode its amino acid sequence was designed and synthesized, and then the gene sequence was constructed on an expression vector to obtain a recombinant plasmid. (2) The recombinant plasmid obtained in step (1) is expressed using the Escherichia coli prokaryotic expression system to obtain the expression product. The glucose dehydrogenase GOX-2015 mutant is obtained by purifying the expression product.

3. A polynucleotide, characterized in that, The polynucleotide encodes the glucose dehydrogenase GOX-2015 mutant as described in claim 1.

4. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression vector capable of correspondingly translating and expressing the glucose dehydrogenase GOX-2015 mutant as described in claim 1.

5. A cell expressing a glucose dehydrogenase GOX-2015 mutant, characterized in that, Escherichia coli BL21(DE3) transformed with the recombinant plasmid as described in claim 4.

6. The application of the glucose dehydrogenase GOX-2015 mutant as described in claim 1 as a catalytic enzyme in the production of NADPH coenzyme by biotransformation.

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