Thermally stable hexokinase mutant and application thereof

Abstract

The invention relates to the field of gene engineering and enzyme engineering, in particular to a thermostable hexokinase mutant and application thereof. The invention provides a hexokinase mutant, the amino acid sequence of the hexokinase mutant is mutated relative to the amino acid sequence of wild type hexokinase, one or more amino acids in the 204 site and the 344 site of the hexokinase mutant are mutated, and the mutated amino acids are selected from lysine, arginine, alanine or leucine; the amino acid sequence of the wild type hexokinase is as shown in SEQ ID NO. 1. The hexokinase mutant provided by the invention can effectively improve the stability of hexokinase from saccharomyces cerevisiae, is beneficial to solving the problem of instability of hexokinase in clinical detection of blood glucose and urine glucose, and has important application value.

Description

Technical Field

[0001] The present application relates to the fields of genetic engineering and enzyme engineering, and in particular to a thermostable hexokinase mutant and its application. Background Art

[0002] Hexokinase is an ATP-dependent, Mg-dependent 2+ It is a predilection alkaline enzyme that plays a key role in sugar metabolism. It catalyzes the phosphorylation of glucose into glucose-6-phosphate, which is the first step of glycolysis. Hexokinase can catalyze the phosphorylation of glucose into glucose-6-phosphate, and in combination with glucose-6-phosphate dehydrogenase, it can convert NAD + Reduced to NADH. The glucose content is quantitatively detected by detecting the change in the absorbance value of NADH at 340nm, thereby realizing the monitoring of blood sugar and urine sugar. This method of detecting blood sugar is one of the internationally recognized standard methods for blood sugar detection: the hexokinase (glucokinase) method. Another standard method for detecting blood sugar is the glucose oxidase method. Compared with this method, the hexokinase method has better specificity and stronger anti-interference ability, and is a more recommended blood sugar detection method in clinical diagnosis. Therefore, hexokinase is also used as a raw material for the development and design of glucose or blood sugar test kits, blood sugar monitoring chips, portable blood sugar detectors and other products. Hexokinase is widely present in various organisms, among which yeast-derived hexokinase is widely used in the IVD field due to its high activity, and is used in blood sugar monitoring and related biochemical analysis.

[0003] A major drawback of yeast-derived hexokinase is its poor thermal stability. The lyophilized preparation of the enzyme needs to be stored at 2-8°C to be stable for 6-12 months. This means that at room temperature or higher, the stability of the enzyme may be reduced, affecting its long-term effectiveness in IVD products. As a result, the costs in the production, transportation, storage and other processes are relatively high and the use environment is restricted, which not only increases the overall cost of blood glucose diagnostic reagents, but also limits its scope of application in different environments. At present, most hexokinase with better stability still relies on imports and is expensive.

[0004] There is an urgent need for highly active and highly thermostable hexokinase in the current market. It is hoped that thermostable hexokinase with higher enzyme activity can be obtained through screening or modification. There are currently three methods to obtain higher activity and better stability: the first is to start with a highly active and less stable hexokinase, such as yeast-derived hexokinase, and to improve its thermostability through random mutation or other modification methods while keeping the activity basically unchanged; the second is to screen hexokinase from different biological sources to obtain enzymes with high activity and good thermostability. The possibility of obtaining stable hexokinase from thermophilic biological sources is greater than that from other biological sources; the third is to increase the thermostability of hexokinase through certain chemical modifications, and the activity remains almost unchanged. This method is used in some imported yeast-derived hexokinase.

[0005] Therefore, it is urgent to solve the problem of poor thermal stability of existing hexokinase so that it can better play a role in practical applications such as sugar metabolism and clinical testing of blood sugar. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, in order to solve the technical problem of poor stability of hexokinase in the prior art, the object of the present invention is to provide a thermostable hexokinase mutant and its use to solve the problems in the prior art.

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

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

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

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

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

[0012] In some embodiments of the present 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] The second aspect of the present application provides an isolated polynucleotide encoding the aforementioned hexokinase mutant.

[0014] The third aspect of the present application provides a construct comprising the aforementioned polynucleotide.

[0015] The fourth aspect of the present application provides a host cell containing the aforementioned construct or the aforementioned polynucleotide integrated into its genome.

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

[0017] The sixth aspect of the present application provides the use of the above-mentioned hexokinase mutant, polynucleotide, construct or host cell in preparing a product.

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

[0019] The seventh aspect of the present application provides a blood sugar or urine sugar detection product, including the above-mentioned hexokinase mutant.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] By implementing random mutation screening on hexokinase, two mutants with stability improvement of more than 50% were found: E204K and T344A; then saturation mutation was performed on these two sites in the hope of obtaining mutants with greater stability improvement, and two mutants, E204R and T344L, were successfully screened out, with stability improvement of more than 60%. These mutants of hexokinase, due to their improved stability, can effectively overcome the instability problems encountered in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The diagram shows the results of the expression and purification of the hexokinase mutants. 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 from top to bottom: 97KD, 66KD, 43KD, 31KD, 20KD, 14KD.

[0023] Figure 2 Shown is a schematic diagram of the stability results for the hexokinase mutants. DETAILED DESCRIPTION

[0024] In order to make the invention purpose, technical scheme and beneficial effect of the present invention clearer, the present invention is further described below in conjunction with examples. It should be understood that the examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified, and those familiar with the technology can easily understand other advantages and effects of the present invention from the contents disclosed in this description.

[0025] After extensive exploration and research, the present application successfully discovered a mutant of hexokinase. Compared with the corresponding wild-type hexokinase, the stability of the mutant is significantly enhanced, thereby enabling it to be better used in the detection of blood sugar and urine sugar. The present invention was completed on this basis.

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

[0027] MVHLGPKKPQARKGSMADVPKELMQQIENFEKIFTVPTETTLQAVTKHFISELEKGLSK

[0028] KGGNIPMIPGWVMDFPTGKESGDFLAIDLGGTNLRVVLVKLGGDRTFDTTQSKYRLPDAM

[0029] RTTQNPDELWEFIADSLKAFIDEQFPQGISEPIPLGFTFSFPASQNKINEGILQRWTKGFDIPNI

[0030] ENHDVVPMLQKQITKRNIPIEVVALINDTTGTLVASYYTDPETKMGVIFGTGVNGAYYDVC

[0031] SDIEKLQGKLSDDIPPSAPMAINCEYGSFDNEHVVLPRTKYDITIDEESPRPGQQTFEKMSSG

[0032] YYLGEILRLALMDMYKQGFIFKNQDLSKFDKPFVMDTSYPARIEEDPFENLEDTDDLFQNE

[0033] FGINTTVQERKLIRRLSELIGARAARLSVCGIAAICQKRGYKTGHIAADGSVYNRYPGFKEK

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

[0035]

[0036] The hexokinase mutant provided in the present application is selected from one of the following situations:

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

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

[0039] In a 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 a 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 a 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 a 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] The present application has obtained four hexokinase mutants through large-scale initial screening of random mutations and further screening of site-directed saturation mutagenesis, which can significantly enhance their stability, with a stability of more than 50%, of which the stability of two hexokinase mutants exceeds 60%. Preferably, the amino acid sequence of the hexokinase mutant is shown in SEQ ID NO.3 or SEQ ID NO.5.

[0048] In the hexokinase mutant provided in the present application, the hexokinase mutant can improve the stability of hexokinase compared with the wild-type hexokinase.

[0049] The hexokinase mutant of the present application has improved stability compared to the wild-type hexokinase; preferably, the stability of the hexokinase derived from Saccharomyces cerevisia is improved. The stability of the hexokinase is represented by the residual enzyme activity, that is, the same hexokinase sample is placed at 4°C and 37°C for 3 consecutive days, and the enzyme activity is determined by the kinetic scanning method.

[0050] Specifically, the hexokinase activity calculation formula is:

[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 calculation formula of the remaining enzyme activity is: Remaining enzyme activity (%) = enzyme activity after treatment at 37° C. for 3 days / enzyme activity after treatment at 4° C. for 3 days×100%.

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

[0055]

[0056]

[0057]

[0058]

[0059] The application also provides a construct, which further comprises additional expression regulatory elements, which are configured to be operably linked to an optional nucleotide sequence. The construct can usually be obtained by inserting the isolated polynucleotide into a suitable vector, and a person skilled in the art can select a suitable vector. For example, the vector can be an expression vector or a cloning vector. For another example, the type of the vector can be a plasmid, a phage, a phage derivative, an animal virus, and a cosmid, etc. Preferably, it is pET39b.

[0060] The present application also provides a host cell, containing the aforementioned construct or the aforementioned polynucleotide integrated with an exogenous source in the genome. The construct is transformed, transduced or transfected into the host cell by conventional methods in the art, such as calcium chloride chemical transformation, high voltage electric shock transformation. Specifically, the host cell can be a prokaryotic cell or a eukaryotic cell, preferably Escherichia coli, Bacillus subtilis, yeast (such as Pichia pastoris, Saccharomyces cerevisiae) or various animal and plant cells, and 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 above-mentioned Escherichia coli needs to be able to express the above-mentioned hexokinase mutant, so as to provide the conditions for the existence of the hexokinase mutant. The method for appropriately constructing the above-mentioned Escherichia coli should be known to those skilled in the art.

[0061] The present application also provides a method for producing hexokinase, comprising the following steps: culturing the aforementioned host cell under the conditions of expressing the aforementioned hexokinase mutant to obtain the aforementioned hexokinase mutant. Suitable induction expression methods should be known to those skilled in the art. For example, the aforementioned host cell can be induced under appropriate conditions to provide hexokinase. For another example, a monoclonal Escherichia coli is inoculated in LB liquid culture medium and cultured at 37°C for 8 to 12 hours; then, at an inoculation ratio of 2 to 5%, it is inoculated into 2xYT culture medium and continued to be cultured at 37°C, and the growth of the cells is continuously monitored. At the ideal stage when the cells grow to the logarithmic growth phase, IPTG is added for induction to promote the expression of hexokinase, and the induction time is controlled at 3 to 12 hours, specifically, it can be 3 to 6 hours, 6 to 8 hours or 8 to 12 hours, etc.

[0062] The present application also provides the use of the aforementioned hexokinase mutant, polynucleotide, construct, or aforementioned host cell in preparing a product. In some embodiments, the product is a product for detecting glucose in vivo, specifically, a product for detecting blood sugar or urine sugar.

[0063] The present application also provides a blood sugar or urine sugar detection product, comprising the above-mentioned hexokinase mutant.

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

[0065] The invention of the present application is further described below by way of examples, but the scope of the present application is not limited thereby.

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

[0067] Example 1

[0068] Random mutagenesis of hexokinase gene and construction of secretory expression vector

[0069] The amplification primers of the hexokinase gene were designed: HK-22b-F / R (Table 1). The coding DNA of hexokinase was obtained by gene synthesis, and then the HK gene was amplified using this as a template. The PCR product was double-digested with BamHI and XhoI, and then constructed into the secretory expression vector pET22b by enzyme digestion and ligation, and sequenced for verification. Then the primers HK-pelB-F / R (Table 1) were designed and the DNA was amplified using QuickMutation TM HK containing the secretion signal peptide pelB was amplified by a gene random mutagenesis kit (Biyuntian, catalog number: D0219S) and random mutagenesis was performed according to the kit instructions. Then, the TA cloning method was directly connected to pBAD TOPOTMTA (Thermo Fisher, catalog number: K430001) to complete the construction and name it: pBAD TOPOTMTA-pelB-HK-Mut.

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

[0071] Primer name Sequence (5'-3') Sequence Listing 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] Large-scale screening of mutants of hexokinase gene

[0074] E. coli Top10 was transformed with pBAD TOPOTMTA-pelB-HK-Mut constructed in Example 1, and then single clones were randomly selected from the plate and inoculated into LB medium containing 500 μl of a final concentration of 0.1% arabinose for overnight culture. After centrifugation, the stability of the mutants was evaluated according to the above-mentioned hexokinase activity and stability detection method, and mutants with stability increased by more than 50% were screened, and DNA sequencing was performed to determine the specific mutation sites that caused the stability change. After screening a large number of mutants, it was found that the mutation of the two amino acids E204K and T344A increased the stability by more than 50% (the stability of the E204K mutant increased by 53%, and the stability of the T344A mutant increased by 57%), thereby determining that E204 and T344 are amino acid positions that have a greater impact on the stability of hexokinase, laying the foundation for the subsequent saturation mutation.

[0075] Example 3

[0076] Saturation mutation of the amino acid positions that affect the stability of hexokinase

[0077] Using the primers HK-pelB-F / R in Example 1, a high-fidelity PCR enzyme was used to amplify HK with a secretion signal peptide pelB, and then constructed into pBAD TOPOTMTA. After successful sequencing and identification, it was named: pBAD TOPOTMTA-pelB-HK. Based on this vector, saturation mutation primers (Table 2) were designed, and saturation mutation was performed using a point mutation kit (Quanshi Jin Biological, catalog number: FM111-01). The activity and stability of the hexokinase mutant were also detected according to the activity and stability detection methods provided above, and the optimal mutations in the two amino acid positions E204 and T344 and the mutations with the most improved stability were screened out and sent to DNA sequencing to determine their specific mutation types. Finally, it was found that E204R and T344L of hexokinase were the optimal mutations in the two amino acid positions, and the stability improvement reached more than 60%.

[0078] Table 2 - Saturation mutagenesis primer sequences

[0079]

[0080]

[0081] Example 4

[0082] Construction of 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 most improved stability as templates. PCR amplification conditions: 98°C for 3 min; 35 cycles (95°C for 20 s, 60°C for 20 s, 72°C for 2 min); 72°C for 10 min. After electrophoresis verification and electrophoresis gel recovery, the purified hexokinase mutant PCR product was obtained. The purified hexokinase mutant PCR product and the pET39b plasmid were double-digested with NdeI and XhoI enzymes, respectively, and then connected with T4 ligase at 16°C for 3 h. The ligation product was transformed into the Escherichia coli Top10 strain and cultured overnight on a solid plate containing LB for colony PCR detection and sequencing verification.

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

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

[0086] Example 5

[0087] Construction and verification of hexokinase mutant expression strain

[0088] The vector successfully constructed in Example 4 was transformed into Escherichia coli BL21 (DE3). Positive monoclones were selected to a final concentration of 50 mg / ml kanamycin in LB medium, and cultured at 37°C until the next day. Subsequently, the bacterial cultures were transferred to new LB culture medium and inoculated at 2%. When the OD600 of the culture medium reached 0.8 to 1.0, IPTG was added thereto at a final concentration of 0.5 mM to induce protein expression, and the culture was continued overnight. Then the cells were broken and the supernatant was taken to measure the crude enzyme activity and verify the expression. Finally, the cells were broken by ultrasound, the supernatant was taken by centrifugation, and a preliminary enzyme activity assay was performed to confirm the successful expression of the protein. On the basis that the amino acid sequence and the coding gene have been disclosed, those skilled in the art can also construct expression vectors of four amino acid mutations of E204K, T344A, E204R, and T344L by synthesizing the corresponding genes, and transform Escherichia coli to express the corresponding mutants.

[0089] Example 6

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

[0091] According to the method described in Example 5, the bacteria after induced expression are collected, and the supernatant is obtained by cell disruption and centrifugation. Since the C-terminus of the expressed hexokinase has a His tag, this property can be used to specifically bind to a nickel metal affinity column to achieve protein purification. During the purification process, the buffers used include: buffer A (25mM Tris-HCL, 150mM NaCl, pH=7.5), and buffer B is based on buffer A with the addition of 500mM imidazole. The target protein was purified by gradient elution using liquid A and liquid B, and a purified protein sample with a molecular weight of 54KD was finally obtained. The results are illustrated using the wild type of hexokinase and two mutants with a stability of more than 60%, as shown in FIG. Figure 1 As shown. The enzyme activity detection of hexokinase refers to the above enzyme activity determination method and calculation formula. The purified HK-WT, HK-E204R and HK-T344L were divided into two parts at the same time, one was placed at 4°C and the other was placed at 37°C for 72 hours to detect the remaining enzyme activity. Then the percentage of remaining enzyme activity was calculated, and the method referred to the above residual enzyme activity calculation formula for hexokinase mutant screening. The results are shown in Figure 2 As shown, the residual enzyme activities of the two hexokinase mutants E204R and T344L were significantly improved compared with the wild type, indicating that the stability of the two mutations E204R and T344L was significantly enhanced.

[0092] Example 7

[0093] Fermentation production of hexokinase

[0094] The constructed hexokinase mutant plasmid was transformed into the Escherichia coli expression strain BL21 (DE3), and the positive single clone was picked and inoculated into LB culture medium, and cultured at 37°C and 200 rpm for 8 hours. Subsequently, the bacterial liquid was inoculated into 2xYT medium at a ratio of 2%, and further cultured using 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, the temperature was adjusted to 30°C, and the expression was induced for 4 to 6 hours.

[0095] In summary, the present invention improves hexokinase by using random mutation and site-directed saturation mutation technology, and successfully screens out two mutants, whose stability is improved by more than 60%. These more stable hexokinase mutants will show better performance in clinical detection of blood sugar and urine sugar.

[0096] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may 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 a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A hexokinase mutant, characterized in that The amino acid sequence of the hexokinase mutant is relative to the amino acid sequence of the wild-type hexokinase, wherein one or more amino acids at positions 204 and 344 are mutated, and the mutated amino acids are selected from lysine, arginine, alanine or leucine; The amino acid sequence of the wild-type hexokinase is shown in SEQ ID NO.

1.

2. The hexokinase mutant according to claim 1, characterized in that 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.

3. The hexokinase mutant according to claim 1, characterized in that The amino acid sequence of the hexokinase mutant is selected from any one of the following: 1) Glutamic acid at position 204 is mutated to lysine or arginine; 2) The threonine at position 344 was mutated to alanine or leucine.

4. The hexokinase mutant according to claim 3, characterized in that 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.

5. An isolated polynucleotide encoding the hexokinase mutant according to any one of claims 1 to 4, preferably, the isolated polynucleotide comprises a sequence as shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO:

10.

6. A construct, characterized in that Contains the polynucleotide as claimed in claim 5.

7. A host cell, characterized in that Contains the construct as claimed in claim 6 or has the polynucleotide as claimed in claim 5 integrated into its genome.

8. A method for producing a hexokinase mutant, comprising culturing the host cell according to claim 7 under conditions suitable for the expression of the hexokinase mutant according to any one of claims 1 to 4 to obtain the hexokinase mutant.

9. Use of the hexokinase mutant according to any one of claims 1 to 4, the nucleotide according to claim 5, the construct according to claim 6 or the host cell according to claim 7 in preparing a product, preferably, the product is a product for detecting blood sugar or urine sugar.

10. A blood sugar or urine sugar detection product, comprising the hexokinase mutant according to any one of claims 1 to 4.

Images