A heat-stable hexokinase mutant and its use

Abstract

The application relates to the fields of genetic engineering and enzyme engineering, and particularly relates to a heat-stable hexokinase mutant and application thereof. The application provides a hexokinase mutant, wherein the amino acid sequence of the hexokinase mutant is mutated at one or more of positions 204 and 344 of the amino acid sequence of wild-type hexokinase, and the mutated amino acid is selected from lysine, arginine, alanine or leucine; and the amino acid sequence of the wild-type hexokinase is shown in SEQ ID NO. 1. The hexokinase mutant provided by the application can effectively improve the stability of hexokinase from Saccharomyces cerevisiae, and is beneficial to solving the instability problem of hexokinase in clinical detection of blood glucose and urine glucose, and has important application value.

Description

Technical Field

[0001] This application relates to the fields of genetic engineering and enzyme engineering, specifically to a thermostable hexokinase mutant and its applications. Background Technology

[0002] Hexokinase is an ATP-dependent, Mg2+ kinase. 2+ Alkaline-biased enzymes play a crucial role in glucose metabolism. They catalyze the phosphorylation of glucose to glucose-6-phosphate, the first step in glycolysis. Hexokinase can catalyze the phosphorylation of glucose to glucose-6-phosphate, and when combined with glucose-6-phosphate dehydrogenase, it can reduce NAD+ levels. + It is reduced to NADH. Glucose content is quantitatively detected by measuring changes in the absorbance of NADH at 340 nm, thus enabling the monitoring of blood glucose and urine glucose. This method of blood glucose detection is one of the internationally recognized standard methods for blood glucose testing: the hexokinase (glucokinase) method. Another standard method for blood glucose detection is the glucose oxidase method. Compared to this method, the hexokinase method has better specificity and stronger anti-interference ability, making it the more recommended blood glucose detection method in clinical diagnosis. Therefore, hexokinase is also used as a raw material in the development and design of glucose or blood glucose test kits, blood glucose monitoring chips, portable blood glucose meters, and other products. Hexokinase is widely found in various organisms, among which yeast-derived hexokinase is widely used in the IVD field due to its high activity, applied to blood glucose monitoring and related biochemical analysis.

[0003] A major drawback of yeast-derived hexokinase is its poor thermostability; its lyophilized formulations require storage at 2-8°C to remain stable for 6-12 months. This means that at room temperature or higher, the enzyme's stability may decrease, affecting its long-term effectiveness in IVD products. This results in relatively high costs during production, transportation, and storage, and restricts its application environment. This not only increases the overall cost of blood glucose diagnostic reagents but also limits their application range in different environments. Currently, most hexokinases with better stability are still imported, making them expensive.

[0004] There is an urgent market demand for highly active and thermostable hexokinases, with a desire to obtain thermostable hexokinases with higher activity than their enzyme activity through screening or modification. Currently, there are roughly three methods to obtain hexokinases with high activity and good stability: First, starting with a highly active but less stable hexokinase, such as yeast-derived hexokinase, and improving its thermostability through random mutation or other modification methods while maintaining its activity essentially unchanged; second, screening hexokinases from different biological sources to obtain enzymes with high activity and good thermostability, with the possibility of obtaining stable hexokinases from thermophilic organisms being greater than from other biological sources; third, increasing the thermostability of hexokinases through certain chemical modifications while maintaining almost no change in activity, a method used in some imported yeast-derived hexokinases.

[0005] Therefore, it is urgent to solve the problem of poor thermal stability of existing hexokinases in order to better play a role in practical applications such as glucose metabolism and clinical blood glucose detection. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, and in order to solve the technical problem of poor stability of hexokinase in the prior art, the purpose of this invention is to provide a thermostable hexokinase mutant and its uses, so as to solve the problems in the prior art.

[0007] To achieve the above and other related objectives, the first aspect of this application provides a hexokinase mutant, wherein the amino acid sequence of the hexokinase mutant is mutated at one or more amino acids at positions 204 and 344 relative to the amino acid sequence of wild-type hexokinase, and the mutated amino acid is selected from lysine, arginine, alanine or leucine; the amino acid sequence of wild-type hexokinase is shown in SEQ ID NO.1.

[0008] In some embodiments of this application, the hexokinase mutant can improve the stability of hexokinase compared to wild-type hexokinase, and preferably can improve the stability of hexokinase derived from Saccharomyces cerevisiae.

[0009] In some embodiments of this application, the amino acid sequence of the hexokinase mutant is selected from any of the following:

[0010] 1) The glutamic acid at position 204 is mutated to lysine or arginine;

[0011] 2) The threonine at position 344 is mutated to alanine or leucine.

[0012] In some embodiments of this application, the amino acid sequence of the hexokinase mutant is shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. Preferably, the amino acid sequence of the hexokinase mutant is shown in SEQ ID NO: 3 or SEQ ID NO: 5.

[0013] A second aspect of this application provides an isolated polynucleotide encoding the aforementioned hexokinase mutant.

[0014] A third aspect of this application provides a construct containing the aforementioned polynucleotides.

[0015] The fourth aspect of this application provides a host cell containing the aforementioned construct or whose genome integrates the aforementioned polynucleotides.

[0016] The fifth aspect of this application provides a method for producing hexokinase, comprising culturing the aforementioned host cells under conditions suitable for the expression of the hexokinase mutant to obtain the hexokinase mutant.

[0017] The sixth aspect of this application provides the use of the above-mentioned hexokinase mutants, polynucleotides, constructs, or host cells in the preparation of products.

[0018] In some embodiments of this application, the product is a product for detecting blood glucose or urine glucose.

[0019] The seventh aspect of this application provides a blood glucose or urine glucose detection product, including the aforementioned hexokinase mutant.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] By conducting random mutation screening of hexokinase, two mutants with stability improvements exceeding 50% were obtained: E204K and T344A. Then, saturation mutations were performed at these two sites to obtain mutants with even greater stability improvements, successfully identifying two mutants, E204R and T344L, both with stability improvements exceeding 60%. These hexokinase mutants, due to their enhanced stability, can effectively overcome the instability issues encountered in practical applications. Attached Figure Description

[0022] Figure 1The diagram shows the purification results of hexokinase mutant expression. Lane 1 is HK-WT supernatant, lane 2 is HK-WT precipitate, lane 3 is HK-WT elution, lane 4 is HK-E204R supernatant, lane 5 is HK-E204R precipitate, lane 6 is HK-E204R elution, lane 7 is HK-T344L supernatant, lane 8 is HK-T344L precipitate, and lane 9 is HK-T344L elution. Lane M is the marker, with values ​​from top to bottom of 97KD, 66KD, 43KD, 31KD, 20KD, and 14KD.

[0023] Figure 2 This diagram shows the stability results of the hexokinase mutant. Detailed Implementation

[0024] To make the inventive objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described below with reference to embodiments. It should be understood that the embodiments are only for explaining the invention and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this invention from the content disclosed in this description.

[0025] Through extensive research and exploration, this application successfully discovered a mutant of hexokinase. Compared with the corresponding wild-type hexokinase, the mutant has significantly enhanced stability, thus enabling its better application in the detection of blood glucose and urine glucose. Based on this, the present invention was completed.

[0026] This application provides a hexokinase mutant, wherein the amino acid sequence of the hexokinase mutant is mutated at one or more amino acids at positions 204 and 344 compared to the amino acid sequence of wild-type hexokinase, and the mutated amino acid is selected from lysine, arginine, alanine, or leucine; the amino acid sequence of wild-type hexokinase is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 6, and the specific sequences are as follows:

[0027] MVHLGPKKPQARKGSMADVPKELMQQIENFEKIFTVPTETTLQAVTKHFISELEKGLSK

[0028] KGGNIPMIPGWVMDFPTGKESGDFLAIDLGGTNLRVVLVKLGGDRTFDTTQSKYRLPDAM

[0029] RTTQNPDELWEFIADSLKAFIDEQFPQGISEPIPLGFTFSFPASQNKINEGILQRWTKGFDIPNI

[0030] ENHDVVPMLQKQITKRNIPIEVVALINDTTGTLVASYYTDPETKMGVIFGTGVNGAYYDVC

[0031] SDIEKLQGKLSDIPPSAPMAINCEYGSFDNEHVVLPRTKYDITIDEESPRPGQQTFEKMSSG

[0032] YYLGEILRLALMDMYKQGFIFKNQDLSKFDKPFVMDTSYPARIEEDPFENLEDTDDLFQNE

[0033] FGINTTVQERKLIRRLSELIGARAARLSVCGIAAICQKRGYKTGHIAADGSVYNRYPGFKEK

[0034] AANALKDIYGWTQTSLDDYPIKIVPAEDGSGAGAAVIAALAQKRIAEGKSVGIIGA (SEQ ID NO. 1).

[0035]

[0036] The hexokinase mutants provided in this application are selected from one of the following:

[0037] 1) The glutamic acid at position 204 is mutated to lysine or arginine;

[0038] 2) The threonine at position 344 is mutated to alanine or leucine.

[0039] In one specific embodiment, when the glutamic acid at position 204 is mutated to lysine, the amino acid sequence of the hexokinase mutant is as shown in SEQ ID NO.2, specifically:

[0040] MVHLGPKKPQARKGSMADVPKELMQQIENFEKIFTVPTETLQAVTKHFISELEKGLSKKGGNIPMIPGWVMDFPTGKESGDFLAIDLGGTNLRVVLVKLGGDRTFDTTQSKYRLPDAMRTTQ NPDELWEFIADSLKAFIDEQFPQGISEPIPLGFTFSFPASQNKINEGILQRWTKGFDIPNIENHDVVPMLQKQITKRNIPIKVVALINDTTGTLVASYYTDPETKMGVIFGTGVNGAYYDVCS DIEKLQGKLSDDIPPSAPMAINCEYGSFDNEHVVLPRTKYDITIDEESPRPGQQTFEKMSSGYYLGEILRLALMDMYKQGFIFKNQDLSKFDKPFVMDTSYPARIEEDPFENLEDTDDLFQN EFGINTTVQERKLIRRLSELIGARAARLSVCGIAAICQKRGYKTGHIAADGSVYNRYPGFKEKAANALKDIYGWTQTSLDDYPIKIVPAEDGSGAGAAVIAALAQKRIAEGKSVGIIGA(SEQ ID NO.2).

[0041] In one specific embodiment, when the glutamic acid at position 204 is mutated to arginine, the amino acid sequence of the hexokinase mutant is as shown in SEQ ID NO.3, specifically:

[0042] MVHLGPKKPQARKGSMADVPKELMQQIENFEKIFTVPTETLQAVTKHFISELEKGLSKKGGNIPMIPGWVMDFPTGKESGDFLAIDLGGTNLRVVLVKLGGDRTFDTTQSKYRLPDAMRTTQ NPDELWEFIADSLKAFIDEQFPQGISEPIPLGFTFSFPASQNKINEGILQRWTKGFDIPNIENHDVVPMLQKQITKRNIPIRVVALINDTTGTLVASYYTDPETKMGVIFGTGVNGAYYDVCS DIEKLQGKLSDDIPPSAPMAINCEYGSFDNEHVVLPRTKYDITIDEESPRPGQQTFEKMSSGYYLGEILRLALMDMYKQGFIFKNQDLSKFDKPFVMDTSYPARIEEDPFENLEDTDDLFQN EFGINTTVQERKLIRRLSELIGARAARLSVCGIAAICQKRGYKTGHIAADGSVYNRYPGFKEKAANALKDIYGWTQTSLDDYPIKIVPAEDGSGAGAAVIAALAQKRIAEGKSVGIIGA(SEQ ID NO.3).

[0043] In one specific embodiment, when the threonine at position 344 is mutated to alanine, the amino acid sequence of the hexokinase mutant is as shown in SEQ ID NO.4, specifically:

[0044] MVHLGPKKPQARKGSMADVPKELMQQIENFEKIFTVPTETLQAVTKHFISELEKGLSKKGGNIPMIPGWVMDFPTGKESGDFLAIDLGGTNLRVVLVKLGGDRTFDTTQSKYRLPDAMRTTQ NPDELWEFIADSLKAFIDEQFPQGISEPIPLGFTFSFPASQNKINEGILQRWTKGFDIPNIENHDVVPMLQKQITKRNIPIEVVALINDTTGTLVASYYTDPETKMGVIFGTGVNGAYYDVCS DIEKLQGKLSDDIPPSAPMAINCEYGSFDNEHVVLPRTKYDITIDEESPRPGQQTFEKMSSGYYLGEILRLALMDMYKQGFIFKNQDLSKFDKPFVMDASYPARIEEDPFENLEDTDDLFQN EFGINTTVQERKLIRRLSELIGARAARLSVCGIAAICQKRGYKTGHIAADGSVYNRYPGFKEKAANALKDIYGWTQTSLDDYPIKIVPAEDGSGAGAAVIAALAQKRIAEGKSVGIIGA(SEQ ID NO.4).

[0045] In one specific embodiment, when the threonine at position 344 is mutated to leucine, the amino acid sequence of the hexokinase mutant is as shown in SEQ ID NO.5, specifically:

[0046] MVHLGPKKPQARKGSMADVPKELMQQIENFEKIFTVPTETLQAVTKHFISELEKGLSKKGGNIPMIPGWVMDFPTGKESGDFLAIDLGGTNLRVVLVKLGGDRTFDTTQSKYRLPDAMRTTQ NPDELWEFIADSLKAFIDEQFPQGISEPIPLGFTFSFPASQNKINEGILQRWTKGFDIPNIENHDVVPMLQKQITKRNIPIEVVALINDTTGTLVASYYTDPETKMGVIFGTGVNGAYYDVCS DIEKLQGKLSDDIPPSAPMAINCEYGSFDNEHVVLPRTKYDITIDEESPRPGQQTFEKMSSGYYLGEILRLALMDMYKQGFIFKNQDLSKFDKPFVMDLSYPARIEEDPFENLEDTDDLFQN EFGINTTVQERKLIRRLSELIGARAARLSVCGIAAICQKRGYKTGHIAADGSVYNRYPGFKEKAANALKDIYGWTQTSLDDYPIKIVPAEDGSGAGAAVIAALAQKRIAEGKSVGIIGA(SEQ ID NO.5).

[0047] This application obtained four hexokinase mutants that significantly enhanced their stability, with a stability exceeding 50%, through large-scale initial screening by random mutation and further screening by site-directed saturation mutation. Among them, two hexokinase mutants had a stability exceeding 60%. Preferably, the amino acid sequence of the hexokinase mutant is shown in SEQ ID NO.3 or SEQ ID NO.5.

[0048] The hexokinase mutant provided in this application improves the stability of hexokinase compared to wild-type hexokinase.

[0049] The hexokinase mutant of this application exhibits improved stability compared to wild-type hexokinase; preferably, the hexokinase derived from *Saccharomyces cerevisiae* shows improved stability. The stability of hexokinase is expressed as residual enzyme activity, i.e., the enzyme activity is determined by placing the same hexokinase sample at 4°C and 37°C for three consecutive days, respectively, and measuring the enzyme activity using a kinetic scan method.

[0050] Specifically, the formula for calculating hexokinase activity is as follows:

[0051]

[0052] Vt: Total reaction volume; Vs: Sample volume; 1.0: Optical path length (cm); 6.22: Molar extinction coefficient of NADH; df: Dilution factor.

[0053] Specifically, the formula for calculating the remaining enzyme activity is: Remaining enzyme activity (%) = Enzyme activity after 3 days of treatment at 37℃ / Enzyme activity after 3 days of treatment at 4℃ × 100%.

[0054] This application also provides an isolated polynucleotide encoding the aforementioned hexokinase mutant. The full-length nucleotide sequence or fragment thereof of the hexokinase mutant of this application can generally be obtained by PCR amplification, recombination, or artificial synthesis. One method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. In a specific embodiment of this application, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0055]

[0056]

[0057]

[0058]

[0059] This application also provides a construct that further includes additional expression regulatory elements configured to be operatively linked to an optional nucleotide sequence. The construct is typically obtained by inserting the isolated polynucleotide into a suitable vector, which can be selected by those skilled in the art. For example, the vector can be an expression vector or a cloning vector. Furthermore, the vector type can be a plasmid, bacteriophage, bacteriophage derivative, animal virus, or entrapment, etc. Preferably, it is pET39b.

[0060] This application also provides a host cell containing the aforementioned construct or whose genome integrates exogenous polynucleotides. The construct is transformed, transduced, or transfected into the host cell using conventional methods in the art, such as chemical transformation using calcium chloride or high-voltage electroporation. Specifically, the host cell can be a prokaryotic or eukaryotic cell, preferably *Escherichia coli*, *Bacillus subtilis*, yeast (such as *Pichia pastoris*, *Saccharomyces cerevisiae*), or various animal or plant cells. More preferably, the host is a genetically engineered bacterium commonly used in the art, such as *Escherichia coli*, *Bacillus subtilis*, *Pichia pastoris*, or *Saccharomyces cerevisiae*. Preferably, the host cell is *Escherichia coli* BL21(DE3) or *Escherichia coli* Top 10. The aforementioned *Escherichia coli* needs to be able to express the aforementioned hexokinase mutant, thereby providing the conditions for the existence of the hexokinase mutant. Suitable methods for constructing the aforementioned *Escherichia coli* should be known to those skilled in the art.

[0061] This application also provides a method for producing hexokinase, comprising the following steps: culturing the aforementioned host cells under the conditions for expressing the hexokinase mutant to obtain the hexokinase mutant. Suitable induction expression methods should be known to those skilled in the art. For example, the aforementioned host cells can be induced under suitable conditions to provide hexokinase. Another example is inoculating a single clone of *E. coli* in LB liquid medium and culturing at 37°C for 8–12 h; then, at an inoculation ratio of 2–5%, inoculating into 2xYT culture medium and continuing to culture at 37°C, while continuously monitoring cell growth. When the cells reach the ideal stage of logarithmic growth, IPTG is added for induction to promote hexokinase expression, with the induction time controlled at 3–12 h, specifically 3–6 h, 6–8 h, or 8–12 h, etc.

[0062] This application also provides the use of the aforementioned hexokinase mutants, polynucleotides, constructs, or host cells in the preparation of the product. In some embodiments, the product is a product for detecting glucose in vivo, specifically, a product for detecting blood glucose or urine glucose.

[0063] This application also provides a blood glucose or urine glucose detection product, including the above-mentioned hexokinase mutant.

[0064] The hexokinase mutant provided by this invention can effectively improve the stability of hexokinase. Experimental data show that, in the presence of the hexokinase mutant of this application, the stability is improved by 60% compared with wild-type hexokinase, which can overcome the stability problem in clinical detection of blood glucose and urine glucose. It also serves as a better raw material for blood glucose and urine glucose test kits or portable point-of-care testing (POCT) devices, thereby making real-time monitoring of blood glucose and urine glucose levels more accurate and stable.

[0065] The invention of this application will be further illustrated by the following embodiments, but this does not limit the scope of this application.

[0066] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0067] Example 1

[0068] Random mutation of the hexokinase gene and construction of a secretory expression vector

[0069] Primers for hexokinase gene amplification were designed: HK-22b-F / R (Table 1). The encoding DNA for hexokinase was obtained through gene synthesis. Using this DNA as a template, the HK gene was amplified. The PCR product was double-digested with BamHI and XhoI, and the resulting DNA was ligated into the secretory expression vector pET22b and sequenced for verification. Next, primers HK-pelB-F / R (Table 1) were designed and used with QuickMutation. TM The random gene mutation kit (Beyotime, catalog number: D0219S) amplified HK containing the secretory signal peptide pelB, and performed random mutations according to the kit instructions. Then, it was directly ligated into pBAD TOPOTMTA (Thermo Fisher, catalog number: K430001) using the TA cloning method to complete the construction, named: pBAD TOPOTMTA-pelB-HK-Mut.

[0070] Table 1 - Primer HK-22b-F / R sequences

[0071] Primer name Sequence (5'-3') Sequence List Number HK-22b-F CGGGATCCGGTTCATTTAGGTCCAAAGAAAACCAC SEQ ID NO:11 HK-22b-R CCGCTCGAGAGCACCGATGATACCAACGG SEQ ID NO:12 HK-pelB-F AGCACCGATGATACCAACGG SEQ ID NO:13 HK-pelB-R ATGAAATACCTGCTGCCGACCGC SEQ ID NO:14

[0072] Example 2

[0073] Extensive screening of mutants of the hexokinase gene

[0074] The pBAD TOPOTMTA-pelB-HK-Mut constructed in Example 1 was transformed into E. coli Top10. Single clones were then randomly selected from the plates and inoculated into LB medium containing 500 μL of 0.1% arabinose for overnight culture. After centrifugation, the stability of the mutants was assessed according to the hexokinase activity and stability detection methods described above. Mutants with stability improvements of more than 50% were screened, and their DNA was sequenced to determine the specific mutation sites leading to the stability changes. After screening a large number of mutants, it was found that the mutations in amino acids E204K and T344A improved stability by more than 50% (E204K mutant showed a 53% increase in stability, and T344A mutant showed a 57% increase). This confirmed that E204 and T344 are the amino acid positions that significantly affect hexokinase stability, laying the foundation for subsequent saturation mutations.

[0075] Example 3

[0076] Saturation mutations were performed on the amino acid positions that affected the stability of hexokinase.

[0077] Using the primers from Example 1: HK-pelB-F / R, HK carrying the secretory signal peptide pelB was amplified using high-fidelity PCR enzyme. This amplified HK was then constructed into pBAD TOPOTMTA, and after successful sequencing identification, it was named pBAD TOPOTMTA-pelB-HK. Based on this vector, saturation mutation primers were designed (Table 2). Saturation mutations were performed using a point mutation kit (TransGen Biotech, catalog number: FM111-01). Following the activity and stability detection methods described above, the activity and stability of the hexokinase mutants were tested. The optimal mutations at the E204 and T344 amino acid positions, as well as the mutations with the greatest stability improvement, were screened and sent for DNA sequencing to determine their specific mutation types. Ultimately, it was found that E204R and T344L were the optimal mutations for the two amino acid positions of hexokinase, with stability improvements exceeding 60%.

[0078] Table 2 - Primer sequences for saturation mutation

[0079]

[0080]

[0081] Example 4

[0082] Constructing intracellular expression vectors for two hexokinase mutants

[0083] Primers HK-F / R (Table 3) were designed, and PCR amplification was performed using the two mutant vectors with the highest stability as templates. PCR amplification conditions: 98℃ for 3 min; 35 cycles (95℃ for 20 s, 60℃ for 20 s, 72℃ for 2 min); 72℃ for 10 min. The purified hexokinase mutant PCR product was obtained after electrophoresis and gel recovery. The purified hexokinase mutant PCR product and pET39b plasmid were double-digested with NdeI and XhoI enzymes, respectively, and then ligated with T4 ligase at 16℃ for 3 h. The ligation product was transformed into E. coli Top10 strains and cultured overnight on LB agar plates for colony PCR detection and sequencing verification.

[0084] Table 3 - Primer HK-F / R sequences

[0085] Primer name Sequence (5'-3') Sequence List Number HK-F GGAATTCCATATGGTTCATTTAGGTCCAAAGAAACCACAA SEQ ID NO:19 HK-R CCGCTCGAGAGCACCGATGATACCAACGGA SEQ ID NO:20

[0086] Example 5

[0087] Construction and validation of hexokinase mutant expression strains

[0088] The vector successfully constructed in Example 4 was transformed into *E. coli* BL21(DE3). Positive single clones were selected and cultured in LB medium with a final concentration of 50 mg / ml kanamycin at 37°C until the next day. These bacterial cultures were then transferred to fresh LB medium and inoculated at 2%. When the OD600 of the culture medium reached 0.8 to 1.0, IPTG at a final concentration of 0.5 mM was added to induce protein expression, and the culture was continued overnight. Cells were then lysed, and the supernatant was collected to measure crude enzyme activity to verify expression. Finally, cells were lysed by sonication, and the supernatant was collected by centrifugation for preliminary enzyme activity assays to confirm successful protein expression. Based on the disclosed amino acid sequences and coding genes, those skilled in the art can also construct expression vectors with four amino acid mutations (E204K, T344A, E204R, and T344L) by synthesizing the corresponding genes and transforming them into *E. coli* to express the corresponding mutants.

[0089] Example 6

[0090] Purification of hexokinase mutants and verification of their activity and stability

[0091] According to the method described in Example 5, bacterial cells after induced expression were collected, and the supernatant was obtained by cell disruption and centrifugation. Since the expressed hexokinase has a His tag at its C-terminus, this property can be utilized to specifically bind to a nickel affinity column, achieving protein purification. The buffers used in the purification process included: Buffer A (25 mM Tris-HCl, 150 mM NaCl, pH = 7.5), and Buffer B, which was Buffer A with the addition of 500 mM imidazole. Gradient elution using solutions A and B was used to purify the target protein, ultimately obtaining a purified protein sample with a molecular weight of 54 kDa. The results are illustrated using wild-type hexokinase and two mutants with stability exceeding 60%, such as... Figure 1 As shown. The enzyme activity of hexokinase was determined according to the enzyme activity assay method and calculation formula described above. Purified HK-WT, HK-E204R, and HK-T344L were simultaneously divided into two portions; one portion was placed at 4°C, and the other at 37°C for 72 hours. The remaining enzyme activity was then measured. The percentage of remaining enzyme activity was calculated using the same formula as described above for calculating remaining enzyme activity during hexokinase mutant screening. The results are shown below. Figure 2 As shown, the residual enzyme activity of both E204R and T344L hexokinase mutants was significantly increased compared with that of wild type, indicating that the stability of the two mutations was significantly enhanced.

[0092] Example 7

[0093] Fermentation production of hexokinase

[0094] The constructed hexokinase mutant plasmid was transformed into Escherichia coli expression strain BL21(DE3). Positive single clones were picked and inoculated into LB medium and cultured at 37°C and 200 rpm for 8 hours. Subsequently, these bacterial cultures were inoculated into 2xYT medium at a ratio of 2% and further cultured in a fermenter. When the OD600 of the culture medium reached the range of 0.8 to 1.0, IPTG was added to a final concentration of 0.5 mM, and the temperature was adjusted to 30°C for induced expression for 4 to 6 hours.

[0095] In summary, this invention has improved hexokinase by employing random mutation and site-directed saturation mutagenesis techniques, successfully screening two mutants with a stability improvement of over 60%. These more stable hexokinase mutants will exhibit superior performance in the clinical detection of blood glucose and urine glucose.

[0096] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A hexokinase mutant, characterized in that, the glutamic acid at position 204 is mutated to lysine or arginine, or the threonine at position 344 is mutated to alanine or leucine, relative to the amino acid sequence of wild-type hexokinase; the amino acid sequence of the wild-type hexokinase is shown as SEQ ID NO.

1.

2. The hexokinase mutant of claim 1, wherein, the amino acid sequence of the hexokinase mutant is shown as SEQ ID NO: 3 or SEQ ID NO:

5.

3. An isolated polynucleotide encoding the hexokinase mutant of claim 1 or 2.

4. The polynucleotide of claim 3, wherein, the sequence of the isolated polynucleotide is shown as SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO:

10.

5. A construct, characterized in that, containing the polynucleotide of claim 3 or 4.

6. A host cell, characterized in that, containing the construct of claim 5 or having integrated in the genome the polynucleotide of claim 3 or 4.

7. A method for producing a hexokinase mutant, comprising culturing the host cell of claim 6 under conditions suitable for expression of the hexokinase mutant of claim 1 or 2 to obtain the hexokinase mutant.

8. Use of the hexokinase mutant of claim 1 or 2, the polynucleotide of claim 3 or 4, the construct of claim 5 or the host cell of claim 6 in the preparation of a product for detecting blood sugar or urine sugar.

9. A product for detecting blood sugar or urine sugar, comprising the hexokinase mutant of claim 1 or 2.

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