Glucose dehydrogenase GOX-2015 mutant and application thereof

Abstract

The invention discloses a glucose dehydrogenase GOX-2015 mutant and application thereof, and relates to the technical field of bioengineering, the amino acid sequence of wild type glucose dehydrogenase GOX-2015 is designed and modified, that is, 18 single mutation sites and 16 combined mutation sites are provided, and the glucose dehydrogenase GOX-2015 mutant is obtained. Compared with the wild type GOX-2015, the thermal stability of the obtained GOX-2015 mutant is improved by about 2 to 13 DEG C, and the activity of the obtained GOX-2015 mutant is improved by about 1.2 to 4.5 times. The invention further provides a preferable mutant protein which has higher yield, activity and thermal stability compared with wild type GOX-2015, has wider application conditions, is more suitable for efficient production of NADPH coenzyme by a biological conversion method, and is beneficial to large-scale production and industrial application.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to a glucose dehydrogenase GOX-2015 mutant and applications thereof. Background Art

[0002] Glucose dehydrogenase (GDH) is an important redox enzyme 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 enzyme thermal stability and catalytic activity have been increasing. Enzymes with high thermal stability can maintain activity in high-temperature environments, making them suitable for high-temperature reaction conditions in industrial production. This also extends the enzyme's lifespan and reduces production costs. Furthermore, high catalytic activity can improve reaction efficiency, reduce enzyme dosage, further reduce costs, and enhance detection sensitivity.

[0004] Although various glucose dehydrogenases have been developed and applied in practical production, they still have certain limitations in terms of thermal stability and catalytic activity. For example, existing GDH mutants, while showing good performance under specific conditions, tend to lose activity under high temperature or prolonged reaction conditions, limiting their wider application.

[0005] Although numerous glucose dehydrogenases have been reported, most exhibit poor thermostability and activity. However, the glucose dehydrogenase GOX-2015, derived from Staphylococcus aureus, has attracted considerable attention due to its higher activity using NADP+ as a coenzyme and superior thermostability compared to other glucose dehydrogenases. A prior study, CN114836396A, disclosed a glucose dehydrogenase mutant, its protein crystals, and applications. This mutant, created by mutating the aspartic acid (D) at position 259 of the wild-type glucose dehydrogenase protein sequence to a cysteine (C), exhibited improved thermostability compared to the wild-type protein. The wild-type protein exhibited a Tm of 64°C, while the mutant exhibited a Tm of 73°C. The D259C mutant also exhibited a yield nearly twice that of the wild-type protein (67.2 mg / L), achieving both improved thermostability and yield. Furthermore, the D259C mutant exhibited 3.5-fold higher NADP+ reduction activity than other reported glucose dehydrogenases. However, the prior study did not compare the activities of the wild-type protein with those of the D259C mutant. In order to further expand the application field of glucose dehydrogenase, it is of great practical significance to develop glucose dehydrogenase mutants with higher thermal stability, catalytic activity and high yield. Summary of the Invention

[0006] The purpose of the present invention is to enable glucose dehydrogenase to meet a wider range of application requirements, especially to be able to be used in high-temperature industrial environments and long-term reaction conditions, and to provide a glucose dehydrogenase GOX-2015 mutant and its application.

[0007] The present invention achieves the above-mentioned purpose through the following technical solutions: As a first aspect of the present invention, a glucose dehydrogenase GOX-2015 mutant is provided, wherein the glucose dehydrogenase GOX-2015 mutant is obtained by subjecting the wild-type GOX-2015 sequence shown in SEQ ID NO.1 to single point mutation or combined mutation; 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 subjecting the wild-type GOX-2015 sequence shown in SEQ ID NO.1 to single point mutation or combined mutation: 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, there is also provided a method for preparing the glucose dehydrogenase GOX-2015 mutant as described above, comprising the following steps: (1) Designing and synthesizing a gene sequence encoding the amino acid sequence of the glucose dehydrogenase GOX-2015 mutant based on its amino acid sequence, and then constructing the gene sequence into an expression vector to obtain a recombinant plasmid; (2) The recombinant plasmid obtained in step (1) is expressed using an Escherichia coli prokaryotic expression system to obtain an expression product, and the expression product is purified to obtain the glucose dehydrogenase GOX-2015 mutant.

[0011] As a third aspect of the present invention, a polynucleotide is also provided, which encodes the glucose dehydrogenase GOX-2015 mutant as described above.

[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, which is an expression vector capable of correspondingly translating and expressing any of the glucose dehydrogenase GOX-2015 mutants 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 above-mentioned recombinant plasmids.

[0016] As a sixth aspect of the present invention, there is provided a use of the glucose dehydrogenase GOX-2015 mutant as described above as a catalytic enzyme in producing NADPH coenzyme using a biotransformation method.

[0017] As a seventh aspect of the present invention, there is provided a use of the glucose dehydrogenase GOX-2015 mutant as described above in improving the activity or thermal stability 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 and combined mutation sites. The resulting GOX-2015 mutants exhibit approximately 2-13°C higher thermal stability and approximately 1.2-4.5-fold higher activity than wild-type GOX-2015. Based on the mutation sites that significantly enhance thermal stability and activity, the invention also provides a preferred GOX-2015 mutant protein (M201K, G115K, H158G, E137S). Compared to wild-type GOX-2015, this mutant protein exhibits higher yield, activity, and thermal stability, broadens its application range, and is more suitable for efficient biotransformation production of NADPH coenzyme, facilitating large-scale production and industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The modified pET-28a vector map provided by the present invention; Figure 2 The results of a small-scale purification of the GOX-2015 single mutant protein provided by the present invention in a cell-free expression system; Figure 3 The results of the small-scale purification of the GOX-2015 combination mutant protein provided by the present invention in a cell-free expression system; Figure 4 Activity test results of the GOX-2015 single mutant protein provided by the present invention; Figure 5 Activity test results of the GOX-2015 combination mutant protein provided by the present invention; Figure 6 The affinity chromatography purification results of the GOX-2015 (M201K, G115K, H158G, E137S) mutant protein provided by the present invention; Figure 7 The quality test results of the GOX-2015 (M201K, G115K, H158G, E137S) mutant protein provided by the present invention; Figure 8 These are the activity test results of the 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 DESCRIPTION

[0020] The present application is described in further detail below in conjunction with the accompanying drawings. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0021] 1. Materials and Reagents The reagents and instruments used in the present invention without manufacturer indication are all conventional products that can be purchased from the market.

[0022] 2. Methods Unless otherwise specified, the methods used in the present invention are conventional methods known to those skilled in the art. If no specific conditions are specified, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

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

[0024] All mutants were constructed based on the wild-type sequence using corresponding mutant primers designed according to molecular cloning methods. A total of 18 single-point mutations and 16 combined mutations were constructed. 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] Combination 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 in a modified pET-28 vector (GenScript). The T7 promoter of this vector was fused with an 8His-strepII-TEV-GG tag sequence. The tag sequence is shown in SEQ ID NO. 2 (wherein, 8His and strepII 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). The gene sequences of the constructed recombinant proteins were verified to be correct by sequencing companies. The vector map is shown in Figure 1 .

[0027] 2.2 Expression and purification of GOX-2015 mutant protein 2.2.1. Small-scale expression of GOX-2015 mutant protein Small-scale 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]. This protocol primarily involves the following two steps: (1) Preparation of E. coli crude extract: Inoculate BL21 (DE3) bacterial suspension into 2×YT medium and culture at 37°C until OD 600 When the OD reaches 0.6-0.8, add isopropylβ-D-1-thiogalactopyranoside (IPTG). 600 When the cell density reaches ≈3, harvest the cells by centrifugation. Rinse each gram of wet 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 cells in 1 mL of S30 buffer per 1 g of cells. Sonicate the cells using an ultrasonic disruptor and centrifuge at 13,000 × g for 10 minutes. Transfer the supernatant to a nuclease-free tube, snap-freeze in liquid nitrogen, and store at -80°C.

[0028] (2) Protein expression in a cell-free system: Plasmid, cell extract, reaction buffer (phosphoenolpyruvate, PEG mixture, potassium glutamate, magnesium glutamate, ammonium glutamate, 20 amino acids, reaction energy substances (nicotinamide adenine dinucleotide, adenosine 5'-triphosphate disodium salt, cytidylic acid disodium, guanosine 5'-monophosphate disodium hydrate and uridylic acid disodium salt, folinic acid calcium salt and tRNA), glucose, spermidine and 1,4-diaminobutane 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°C, 200 rpm. After 4 hours, the reaction solution was centrifuged at 12,000 rpm for 10 minutes, the supernatant and precipitate were collected, and samples of each step were fixed by loading.

[0029] 2.2.2. Small-scale purification of GOX-2015 mutant protein (1) Small-scale purification of GOX-2015 single mutant protein The supernatant after expression in the cell-free system was added to 50 μL Strep-Tactin®XT filler treated with buffer (25 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM TCEP), and incubated at 4°C for 30 minutes. The incubated sample was centrifuged at 12,000 rpm at 4°C for 10 minutes, 1 mL of buffer was added, and after washing three times, 100 μL of elution buffer (25 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM TCEP, 75 mM biotin) was added. The sample was centrifuged at 12,000 rpm at 4°C for 5 minutes, and the eluted sample was collected. A small amount of sample was retained for fixation with loading buffer and detected by SDS-PAGE.

[0030] The experimental results are shown in Figure 2 Among the 18 single-point mutations, all single-point mutant proteins were significantly expressed, and their aggregation states were consistent with the wild-type protein, which was a tetrameric protein.

[0031] (2) Small-scale purification of GOX-2015 combination mutant proteins The purification method for the combined mutants was the same as that for the single-point mutant proteins. Pilot purification results showed that all 16 combined mutant proteins were clearly expressed in the cell-free system. FSEC analysis of the expressed samples revealed that all the combined mutant proteins aggregated as tetramers. All expressed samples were further purified.

[0032] The purification results are shown in Figure 3 , all samples can be eluted with high purity.

[0033] 2.3. Thermal stability test of GOX-2015 mutant protein The thermal stability of the GOX-2015 mutant protein was tested using the Protein Thermal Shift (ThermoFluor) technique. This technique exploits the structural characteristics of proteins: proteins have hidden hydrophobic regions. When the temperature rises, these regions open up, exposing the hydrophobic regions. The fluorescent dye SYPRO Orange binds to these regions and excites them to fluoresce. The change in fluorescence signal intensity forms a melting curve, and the temperature corresponding to the maximum value of the melting curve derivative is the melting point (Tm). The more stable the protein, the higher the measured Tm value.

[0034] The specific operations are as follows: 5 μg of each GOX-2015 mutant protein was added to a 96-well PCR plate. 10× SYPRO Orange fluorescent dye was then added to the corresponding wells. The 96-well PCR plate was placed in a qPCR instrument. The instrument parameters were set and the temperature was increased from 25°C to 99°C in a gradient of 1°C per minute. The protein melting curve was calculated. The specific numerical values of the T values determined for all single mutant proteins are shown in Table 1.

[0035] Table 1 Tm values of GOX-2015 single mutant proteins ; As shown in Table 1, among the 18 single mutant proteins, only the E62I and K77S mutations had a slight increase in Tm values, while the others all increased by at least 2°C. Among them, the Tm values of G115A, G115K, G115R, H158G, D230A, S225P, S265L, N263P, N263D, and S265F increased by 3-5°C. Therefore, the present invention screened out 16 mutants, including 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. Moreover, judging from the Tm value data, the amino acids G115, M201, N263 and S265 play an important role in protein thermal stability.

[0036] The single mutation sites M201K, G115K, G115A, G115R, H158G, D230A and S225P with relatively obvious improvement were selected for combination. After small-scale expression and purification, the Tm values were tested. The test results are shown in Table 2.

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

[0038] In addition, the Tm values of 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. GOX-2015 mutant protein activity detection To further verify the activity of the mutant proteins with improved thermal stability, activity tests 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 detection system provided by the present invention uses glucose as a substrate, adds the coenzyme NADP+, and determines the enzymatic activity of different GOX-2015 mutant proteins by detecting the fluorescence absorption signal of NADPH generated by the reaction at 340nm.

[0040] The specific experimental operations are as follows: (1) Prepare the buffer solution: 50 mM Tris pH 8.0, 5 mM glucose as substrate, 2 mM NADP+ as coenzyme, and a reaction temperature of 20°C. Transfer 30 μL of substrate and coenzyme NADP+ to a 384-well plate and set up two replicate wells. Transfer 50 nM of the GOX-2015 mutant protein to be tested to the corresponding wells of the plate. Immediately centrifuge and shake to mix. Use a TECAN F200 microplate reader to collect the fluorescence signal generated by the reaction. Use GraphPad Prism 9 analysis software for data analysis to obtain the enzyme activity parameters of the tested protease.

[0041] (2) Analysis of results: According to the results of enzyme activity assay, 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, and N263P, N263D, S265L, and S265F were basically inactive. The activities of other mutant proteins were maintained between 50% and 75% of the wild-type protein activity ( Figure 4 ).

[0042] Combined mutant proteins (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) A total of 5 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, the activities of (M201K, H158G), (M201K, G115R), (M201K, G115K), (M201K, G115K, H158G, E137S) and (M201K, G115K, H158G, E62I) were 1.2-1.7 times higher than that of the wild-type ( Figure 5 ). From these data, it can be seen that D259C has a significant effect on the activity of GOX-2015 protein, and the activities of mutants without D259C mutation are higher than those with D259C mutation.

[0043] Combined with the data results of thermal stability and activity, it can be seen that among the single mutations, M201K, E62I, K77S, E137S, and D205N are high-quality mutation sites with improved thermal stability and activity; among the combined mutations, (M201K, H158G), (M201K, G115R), (M201K, G115K), (M201K, G115K, H158G, E137S) and (M201K, G115K, H158G, E62I) are mutant proteins with improved thermal stability and activity.

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

[0045] 2.5.1. GOX-2015 (M201K, G115K, H158G, E137S) mutant protein expression After the recombinant plasmid of the GOX-2015 (M201K, G115K, H158G, E137S) protein was transformed into BL21 (DE3) strain, the strain was inoculated into 50 ml LB liquid medium and cultured at 37 ° C overnight. The overnight cultured bacteria were inoculated into 1 L LB liquid medium at a ratio of 1:100 and cultured at 37 ° C until the bacterial solution OD 600 When the pH value was 0.6-0.8, 0.5 mM IPTG was added, cultured overnight at 15°C, and the cells were collected by centrifugation at 5000 rpm for purification.

[0046] 2.5.2 Purification of GOX-2015 (M201K, G115K, H158G, E137S) mutant protein (1) Affinity chromatography The collected bacterial blocks were weighed separately, and the corresponding volume of lysis buffer (50mM Tris-HCl (pH7.5), 500mM NaCl, 5% glycerol) was added at a ratio of 1:10. The bacteria were broken up using a high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation at 16000rpm. The protein was enriched and purified using a Ni Bestarose FF affinity chromatography column. The specific process is as follows: first, the Ni Bestarose FF affinity chromatography column was washed and balanced with lysis buffer for 10 column volumes, and then the lysis supernatant was loaded onto the Ni Bestarose FF affinity chromatography column, and eluted with lysis buffer containing 50mM imidazole. The eluted protein was collected for SDS-PAGE detection, and the protein concentration was measured with Nanodrop to calculate the protein yield. The protein purification results are shown in Figure 2. Figure 6 , with a yield of 100 mg / L. Previous studies have shown that the yield of the wild-type protein is approximately 67.2 mg / L. It can be seen that the yield of the GOX-2015 (M201K, G115K, H158G, E137S) mutant protein is nearly 1.5 times higher than that of the wild-type.

[0047] (2) Enzyme digestion and reverse affinity chromatography To obtain a higher purity protein, a certain amount of TEV enzyme (Biortus, BP11748) was added to the sample after the above affinity chromatography. After enzymatic digestion at 4°C overnight, the supernatant was further purified using a HisFF chromatography column. Because GOX-2015 (M201K, G115K, H158G, E137S) has no affinity tag after enzymatic digestion, it will not bind to the affinity column and will flow out of the column (recorded as the flowthrough), so the flowthrough was collected.

[0048] (3) Gel filtration chromatography and QC testing The above-mentioned flowthrough was concentrated to about 2 mL and then subjected to gel filtration chromatography. The gel chromatography column model was: Superdex 200 Increase 10 / 300GL, and the gel chromatography buffer was: 25mM HEPES (pH 7.5), 100mM NaCl. The samples after gel filtration chromatography were collected and protein quality was tested, namely SDS-PAGE purity test, mass spectrometry analysis and analytical molecular sieve test. The test results were as follows: Figure 7 shown.

[0049] SDS-PAGE results showed that the purity of the wild-type and GOX-2015 mutant proteins was greater than 99%. Mass spectrometry also showed that the molecular weight of the test samples was essentially consistent with that of the target protein, confirming that the purified proteins were indeed the target proteins. Furthermore, analytical molecular sieve analysis revealed that all proteins were nearly tetrameric in solution.

[0050] 2.5.3. Activity assay of GOX-2015 (M201K, G115K, H158G, E137S) mutants After obtaining a highly pure GOX-2015 (M201K, G115K, H158G, E137S) mutant, its activity was compared with that of wild-type GOX-2015 (Biortus, BP15508-00A) and the D259C mutant protein (Biortus, BP17903-00A) from a previous study. The activity detection method remained consistent with that used in the previous study.

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

[0052] 3. Conclusion The GOX-2015 mutant protein provided by the present 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 above-described embodiments merely illustrate several implementations of the present invention. 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 a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements 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 is obtained by subjecting the wild-type GOX-2015 sequence shown in SEQ ID NO.1 to single-point mutation or combined mutation; 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.

2. A glucose dehydrogenase GOX-2015 mutant according to claim 1, characterized in that The glucose dehydrogenase GOX-2015 mutant is obtained by subjecting the wild-type GOX-2015 sequence shown in SEQ ID NO.1 to single point mutation or combined mutation: 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.

3. A glucose dehydrogenase GOX-2015 mutant according to claim 2, characterized in 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.

4. A method for preparing the glucose dehydrogenase GOX-2015 mutant according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Designing and synthesizing a gene sequence encoding the amino acid sequence of the glucose dehydrogenase GOX-2015 mutant based on its amino acid sequence, and then constructing the gene sequence into an expression vector to obtain a recombinant plasmid; (2) The recombinant plasmid obtained in step (1) is expressed using an Escherichia coli prokaryotic expression system to obtain an expression product, and the expression product is purified to obtain the glucose dehydrogenase GOX-2015 mutant.

5. A polynucleotide, characterized in that The polynucleotide encodes the glucose dehydrogenase GOX-2015 mutant according to any one of claims 1 to 3.

6. A polynucleotide according to claim 5, characterized in that The polynucleotide sequence encoding the amino acid sequence of the glucose dehydrogenase GOX-2015 mutant shown in SEQ ID NO.3 is shown in SEQ ID NO.

4.

7. 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 according to any one of claims 1 to 3.

8. A glucose dehydrogenase GOX-2015 mutant expression system, characterized in that: The recombinant plasmid according to claim 7 is transformed into Escherichia coli BL21 (DE3).

9. Use of the glucose dehydrogenase GOX-2015 mutant according to any one of claims 1 to 3 as a catalytic enzyme in producing NADPH coenzyme using a bioconversion method.

10. Use of the glucose dehydrogenase GOX-2015 mutant according to any one of claims 1 to 3 in improving the activity or thermal stability of glucose dehydrogenase.

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