A hexokinase, its preparation method and application

Abstract

The present invention provides a hexokinase, a preparation method thereof and an application. Among them, the hexokinase is obtained by mutating the hexokinase gene of wild-type Saccharomyces cerevisiae; the 52nd position of the amino acid sequence of the hexokinase is replaced and modified to E52V, and the 254th position is replaced and modified to S254P; the amino acid sequence is as shown in SEQ ID N0.1. The technical problem solved by the present invention is that the cost of the kit is high and the stability is poor during the detection of glucose by the hexokinase method.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular, to a hexokinase, a preparation method thereof, and an application thereof. Background Art

[0002] Glucose detection is a commonly used biochemical detection item, and it is widely used in the diagnosis of diabetes, the observation of curative effects, and general clinical diagnosis and treatment. At present, the mainstream method for detecting glucose clinically is by enzymatic method, among which the commonly used ones are glucose oxidase method and hexokinase method. The glucose oxidase method is easily interfered by reducing substances such as bilirubin and ascorbic acid, etc. The hexokinase method has high specificity for glucose and is not interfered by bilirubin, etc., but the related enzymes required for its reaction have poor stability.

[0003] Hexokinase, as a key raw material in the glucose detection kit, the current preparation method is mainly to extract it from natural bacteria such as yeast. Its extraction process is cumbersome, the yield is low, the production cost is high, and the natural enzyme has poor thermal stability. These lead to high cost of the kit on the one hand and poor reagent stability on the other hand, restricting the clinical application of the kit. The high cost and poor stability of the kit in the process of detecting glucose by the hexokinase method are problems that need to be urgently solved in clinical diagnosis. Summary of the Invention

[0004] The technical problem solved by the present invention is the high cost and poor stability of the kit in the process of detecting glucose by the hexokinase method.

[0005] To solve the above problems, the present invention provides a hexokinase, which is obtained by mutating the hexokinase gene of wild-type Saccharomyces cerevisiae; the 52nd position of the amino acid sequence of the hexokinase is replaced and modified to E52V, and the 254th position is replaced and modified to S254P; the amino acid sequence is as shown in SEQ ID N0.1.

[0006] Compared with the prior art, the technical effects achieved by adopting this technical solution: the hexokinase in the present invention is obtained by mutating the hexokinase gene of wild-type Saccharomyces cerevisiae, and two amino acids in the amino acid sequence of the hexokinase mutate, one is that the 52nd position is replaced and modified to E52V, and the other is that the 254th position is replaced and modified to S254P; realizing the production of a recombinant hexokinase with high catalytic activity and good thermal stability, thereby improving the glucose detection reagent by the hexokinase method and improving its clinical detection efficiency.

[0007] Furthermore, the present invention also provides a method for preparing hexokinase, comprising the following steps: Step 1: Using the hexokinase gene sequence of wild-type Saccharomyces cerevisiae as a template, performing error-prone PCR amplification mutagenesis to prepare a gene random mutation library; Step 2: Using the genes in the random mutation library for recombinant expression; Step 3: Preliminary screening of mutant strains, selecting 10 hexokinase mutant strains with improved thermal stability; Step 4: Re-screening of mutant strains, selecting 1 hexokinase mutant strain with high catalytic activity and high thermal stability.

[0008] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: By using the wild-type hexokinase gene sequence of Saccharomyces cerevisiae as a template, performing error-prone PCR random mutagenesis to construct a hexokinase mutation library, through preliminary screening of mutant strains, 10 hexokinase mutant strains with improved thermal stability are selected, and through re-screening of mutant strains, finally 1 hexokinase with high catalytic activity and good thermal stability and its recombinant expression strain are selected. The amino acid sequence of the hexokinase expressed by this recombinant expression strain is shown in SEQ ID N0.1.

[0009] Furthermore, Step 2 includes: Digesting the gene with a specific endonuclease and ligating it to an expression vector, and the expression vector contains a His-tag label.

[0010] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: The expression vector contains a His-tag label, which enables the rapid and efficient purification of hexokinase by Ni affinity chromatography.

[0011] Furthermore, Step 2 includes: Transforming and introducing the expression vector into a genetically engineered bacterium for recombinant expression, and the genetically engineered bacterium includes Escherichia coli.

[0012] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: By transforming and introducing the expression vector into a genetically engineered bacterium for recombinant expression, where the genetically engineered bacterium includes Escherichia coli. Hexokinase can be recombinantly expressed and produced by the Escherichia coli genetically engineered bacterium. The culture method of Escherichia coli is simple, the cost of the required culture medium is low, and the expression level is high. By ligating hexokinase to an expression vector containing a label and then transforming and introducing the expression vector into Escherichia coli, the purification is simple and the recovery rate is high, and the production cost of hexokinase is low, and industrial large-scale production can be achieved.

[0013] Furthermore, the preliminary screening of mutant strains in Step 3 includes: Randomly picking multiple single colonies from the recombinant expression plate, culturing and expressing them, and calculating the enzyme retention rate through direct cell lysis and heat treatment.

[0014] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: Through the primary screening of mutant strains, multiple single colonies are randomly picked from the recombinant expression plate for culturing and expression. By directly lysing the cells and performing heat treatment, the enzyme retention rate is calculated. Ten hexokinase mutant strains with improved thermal stability are selected.

[0015] Furthermore, the rescreening of mutant strains in step four includes: performing shake-flask expression, purification, and recovery of the enzyme solution on the ten hexokinase mutant strains with improved thermal stability, measuring the enzyme activity and protein content of the enzyme solution, and calculating the specific enzyme activity.

[0016] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: Through the rescreening of mutant strains, shake-flask expression, purification, and recovery of the enzyme solution are performed on the ten hexokinase mutant strains with improved thermal stability. The enzyme activity and protein content of the enzyme solution are measured, and the specific enzyme activity is calculated. Finally, one hexokinase mutant strain with high catalytic activity and high thermal stability is preferably selected.

[0017] Furthermore, for the recombinant expression gene engineering bacteria of hexokinase mutant strains in step four, a sequencing reaction is performed on the DNA sequence of the gene engineering bacteria to obtain the nucleotide coding sequence of hexokinase.

[0018] Furthermore, the nucleotide coding sequence of hexokinase is as shown in SEQ ID N0.2; or the nucleotide coding sequence of hexokinase includes a nucleotide sequence obtained by one or several nucleotide substitutions of the nucleotide sequence shown in SEQ ID N0.2, which is a synonymous mutation of the codon.

[0019] Furthermore, compared with the original gene sequence, three base sequences of the nucleotide sequence SEQ ID N0.2 have changed. Specifically, at position 155, A changes to T; at position 760, T changes to C; at position 846, A changes to T.

[0020] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: Through the DNA sequencing reaction, the nucleotide coding sequence of this hexokinase is as shown in SEQ ID N0.2. Compared with the original gene sequence, three nucleotide sequences have mutated, namely at position 155, A→T; at position 760, T→C; at position 846, A→T. Among them, the mutation at position 846 is a synonymous base mutation, which does not cause a change in the encoded amino acid; the base mutation at position 155 causes the original codon GAA encoding Glu to mutate into GUA encoding Val; the base mutation at position 760 causes the original codon UCA encoding Ser to mutate into CCA encoding Pro.

[0021] The present invention also provides an application of hexokinase, applying hexokinase to a glucose detection kit.

[0022] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: The hexokinase provided by the present invention is applied to an in vitro glucose detection kit. When used for glucose detection, the performance of this kit not only meets the clinical requirements, such as anti-interference, accuracy, precision, linear range, reagent blank, etc., but also the enzyme has high catalytic activity, which can reduce the amount of enzyme used and lower the reagent cost. This enzyme is produced by genetically engineered bacteria, further reducing the reagent production cost; this hexokinase has good thermal stability, which can improve the reagent stability. After the reagent is stored at 37°C for 11 days under accelerated conditions and stored at 4°C for 18 months, the performance indicators of the kit are all good.

[0023] After adopting the technical solution of the present invention, the following technical effects can be achieved:

[0024] (1) The hexokinase obtained in the present invention has high catalytic activity and good thermal stability. The specific enzyme activity of its catalytic reaction of glucose with adenosine triphosphate is 630 U / mg. After being treated at 55°C for 30 min, the enzyme activity retention rate is still 62.1%;

[0025] (2) The hexokinase obtained in the present invention can be recombinantly expressed and produced by genetically engineered Escherichia coli. The cultivation method of Escherichia coli is simple, the cost of the required culture medium is low, the expression level is high, the purification with a tag is simple, and the recovery rate is high, making the production cost of this enzyme low and enabling industrial large-scale production;

[0026] (3) The hexokinase obtained in the present invention is applied to an in vitro glucose detection kit. When used for glucose detection, the performance of this kit not only meets the clinical requirements, such as accuracy, precision, anti-interference, linear range, reagent blank, etc., but also the enzyme has high catalytic activity, which can reduce the amount of enzyme used and lower the reagent cost. This enzyme can be produced by genetically engineered bacteria, further reducing the reagent production cost. The obtained hexokinase has good thermal stability, which can improve the reagent stability. After the reagent is stored at 37°C for 11 days under accelerated conditions and stored at 4°C for 18 months, the performance indicators of the kit are all good. Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings to be used in the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings;

[0028] Att Figure 1 : Enzyme activity retention rate of hexokinase at different temperatures;

[0029] Att Figure 2 : Linear relationship before and after heat storage of HK applied to glucose detection kit. Detailed Embodiments

[0030] To make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of specific embodiments of the present invention will be given in conjunction with the accompanying drawings.

[0031] The present invention provides a hexokinase, which is obtained by mutating the hexokinase gene of wild-type Saccharomyces cerevisiae; the 52nd position of the amino acid sequence of the hexokinase is replaced and modified to E52V, and the 254th position is replaced and modified to S254P; the amino acid sequence is as shown in SEQ ID N0.1.

[0032] In this embodiment, the hexokinase is obtained by mutating the hexokinase gene of wild-type Saccharomyces cerevisiae. Two amino acids on the sequence of this enzyme are mutated, namely E52V and S254P; the production of a recombinant hexokinase with high catalytic activity and good thermal stability is realized, thereby improving the hexokinase method glucose detection reagent and enhancing its clinical detection efficiency.

[0033] The present invention also provides a method for preparing hexokinase, which includes the following steps: Step 1: Using the hexokinase gene sequence of wild-type Saccharomyces cerevisiae as a template, performing error-prone PCR amplification mutation to prepare a gene random mutation library; Step 2: Using the genes in the random mutation library for recombinant expression; Step 3: Preliminary screening of mutant strains, selecting 10 hexokinase mutant strains with improved thermal stability; Step 4: Re-screening of mutant strains, selecting 1 hexokinase mutant strain with high catalytic activity and high thermal stability.

[0034] In a specific embodiment of the present invention, using the hexokinase HKII gene sequence of Saccharomyces cerevisiae as a template, error-prone PCR amplification mutation is carried out to establish a random mutation library. By using the wild-type hexokinase gene sequence of Saccharomyces cerevisiae as a template, error-prone PCR random mutation is performed to construct a hexokinase mutation library. After preliminary screening of mutant strains, 10 hexokinase mutant strains with improved thermal stability are selected. After re-screening of mutant strains, finally 1 hexokinase with high catalytic activity and good thermal stability and its recombinant expression strain are selected. The amino acid sequence of the hexokinase expressed by this recombinant expression strain is as shown in SEQ ID N0.1.

[0035] Further, Step 2 includes: digesting the gene with a specific endonuclease and ligating it to an expression vector, and the expression vector contains a His-tag label.

[0036] In a specific embodiment of the present invention, using the genes in the random mutation library, through digestion with a specific endonuclease and ligation with a ligase to the PET28 vector, the expression vector contains a His-tag label, which can enable the hexokinase to be rapidly and efficiently purified by Ni affinity chromatography.

[0037] Further, step two includes: transforming and introducing the expression vector into a genetically engineered bacterium for recombinant expression. The genetically engineered bacterium includes Escherichia coli.

[0038] In a specific embodiment of the present invention: The expression vector is transformed and introduced into the genetically engineered bacterium E. coli BL21(DE32) for recombinant expression. Hexokinase can be produced by recombinant expression in Escherichia coli genetically engineered bacteria. The culture method of Escherichia coli is simple, the cost of the required culture medium is low, and the expression level is high. At the same time, hexokinase is linked to an expression vector containing a tag, and then the expression vector is transformed and introduced into Escherichia coli, making the purification simple and the recovery rate high. The production cost of hexokinase is low, and industrial large-scale production can be achieved.

[0039] Further, the primary screening of mutant strains in step three includes: randomly picking multiple single colonies from the recombinant expression plate, culturing and expressing them, and calculating the enzyme retention rate through direct cell lysis and heat treatment.

[0040] In a specific embodiment of the present invention: For the primary screening of mutant strains in centrifuge tubes, approximately 100 single colonies are randomly picked from the recombinant expression plates with a large number of random mutations, cultured and expressed in centrifuge tubes, and the enzyme retention rate is calculated through direct cell lysis and heat treatment. 10 hexokinase mutant strains with improved thermal stability are selected.

[0041] Further, the secondary screening of mutant strains in step four includes: performing shake-flask expression, purification, and recovery of the enzyme solution on 10 hexokinase mutant strains with improved thermal stability, and performing enzyme activity determination and protein determination on the enzyme solution to calculate the specific enzyme activity.

[0042] In a specific embodiment of the present invention: For the secondary screening of mutant strains in shake flasks, 10 hexokinase mutant strains with improved thermal stability are subjected to shake-flask expression, purification, and recovery of the enzyme solution. Enzyme activity determination and protein determination are performed on the enzyme solution to calculate the specific enzyme activity, and finally 1 hexokinase mutant strain with high catalytic activity and high thermal stability is preferably selected.

[0043] Further, for the genetically engineered bacterium of the hexokinase mutant strain in step four, a sequencing reaction is performed on the DNA sequence of the genetically engineered bacterium to obtain the nucleotide coding sequence of hexokinase.

[0044] In a specific embodiment of the present invention: For the preferably selected 1 hexokinase mutant recombinant expression genetically engineered bacterium, recombinant expression is performed again, the enzyme solution is purified and recovered, and its specific enzyme activity and thermal stability are further determined. The specific enzyme activity of the reaction catalyzed by glucose and adenosine triphosphate is measured to be 630 U / mg. After the enzyme solution is treated at 55 °C for 30 min, the enzyme activity retention rate can reach 62.1%. A sequencing reaction is performed on the DNA sequence of the recombinant expression genetically engineered bacterium.

[0045] Furthermore, the hexokinase nucleotide coding sequence is as shown in SEQ ID NO.2; or the hexokinase nucleotide coding sequence includes a nucleotide sequence with codon synonymous mutations obtained by substituting one or several nucleotides in the nucleotide sequence shown in SEQ ID NO.2.

[0046] Furthermore, compared with the original gene sequence, the nucleotide sequence of SEQ ID NO.2 has three base sequence changes, specifically at position 155, A changes to T; at position 760, T changes to C; at position 846, A changes to T.

[0047] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: Through DNA sequencing reaction, the hexokinase nucleotide coding sequence is as shown in SEQ ID NO.2. Compared with the original gene sequence, there are 3 nucleotide sequence mutations, which are at position 155, A→T; at position 760, T→C; at position 846, A→T. Among them, the mutation at position 846 is a synonymous base mutation and does not cause a change in the encoded amino acid; the base mutation at position 155 causes the original codon GAA encoding Glu to mutate into GUA encoding Val; the base mutation at position 760 causes the original codon UCA encoding Ser to mutate into CCA encoding Pro.

[0048] The present invention also provides an application of hexokinase, applying hexokinase to a glucose detection kit.

[0049] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: The hexokinase provided by the present invention is applied to an in vitro glucose detection kit. For glucose detection by this kit, not only the reagent performance meets clinical requirements, such as anti-interference, accuracy, precision, linear range, reagent blank, etc., but also the catalytic activity of this enzyme is high, which can reduce the enzyme dosage and lower the reagent cost. This enzyme is produced by genetically engineered bacteria, further reducing the reagent production cost; the obtained hexokinase has good thermal stability, which can improve the reagent stability. When the reagent is stored at 37°C for 11 days under accelerated heat storage and stored at 4°C for 18 months, the performance indicators of the kit are all good.

[0050] Example 1

[0051] Error-prone PCR amplification of the hexokinase gene sequence to prepare a gene random mutation library

[0052] According to the HK sequence of Saccharomyces cerevisiae, primer sequences are designed:

[0053] Forward primer: 5’-CAAGTAAACCCAGGTTTTTT-3’,

[0054] Reverse primer: 5’-TCAAGCACCAATAATACCAA-3’

[0055] Using the above primers, error-prone PCR amplification was carried out. The error-prone PCR amplification system was as follows:

[0056] 10× amplification buffer 30 μl

[0057] dNTP mixture 40 μM

[0058] Primer 70 pM

[0059] Template 0.6 μg

[0060] Error-prone Taq DNA polymerase 3 U / L

[0061] Mg 2+ 0.2 mM

[0062] Mn 2+ 8 mM

[0063] Add double-distilled water to 250 μl.

[0064] The PCR reaction conditions were: 92°C for 5 min; 92°C for 30 s, 55°C for 30 s, 72°C for 2 min, 35 cycles; 72°C for 10 min, 4°C.

[0065] Take 6 μl of the above PCR amplification product for agarose gel electrophoresis. The target product band was observed at about 1500 bp, and it was determined that the PCR amplification band was obtained. After the PCR amplification product was purified and recovered by a DNA recovery kit, a large number of randomly mutated gene fragments were obtained. They were digested with NdeI and XhoI enzymes and ligated into PET28 with T4 ligase. Then they were transformed into the E. coli BL21(DE32) expression strain and spread on an LB plate containing 60 mg / L ampicillin (AMP). They were cultured overnight at 37°C to obtain a large number of single colonies of genetically engineered bacteria containing randomly mutated gene fragments for subsequent screening.

[0066] Example 2

[0067] Preliminary screening of mutant strain centrifuge tubes

[0068] Pick the monoclonal strains on the LB plate of the large number of randomly mutated expression strains obtained in Example 1 into 10 ml of LB liquid medium. Culture at 37°C until the OD is about 0.6, add IPTG with a final concentration of 0.1 mM, and cool down to 20°C for induction expression for more than 16 h.

[0069] Centrifuge the culture medium at 3000 rpm for 15 min, discard the supernatant and retain the cells. Add 5 ml of cell lysate, the composition of which is 20 mM Tris-HCl, 0.8 g / L CTAB, 20 mM MgCl2, 0.5 g / L sodium deoxycholate, pH 7.0, and lyse for 30 min to obtain the crude enzyme extract. Then place the crude enzyme extract in an incubator at 55 °C for about 30 min.

[0070] Take 10 μl of each crude enzyme extract and add it to 1 ml of enzyme activity reaction solution, continuously detect the change rate of absorbance at 340 nm for 2 min at 37 °C. The composition of the enzyme activity reaction solution is: 50 mM Tris-HCl pH 8.0, ATP 2 mM, NAD + 5 mM, MgCl2 10 mM, glucose-6-phosphate dehydrogenase 10 U / ml, Glu 0.2 g / L. The principle of the activity measurement reaction is: hexokinase, under the activation of Mg 2+ catalyzes the reaction of Glu and ATP to generate glucose-6-phosphate, and glucose-6-phosphate further undergoes a dehydrogenation reaction under the action of glucose-6-phosphate dehydrogenase and NAD + to produce NADH. NADH has an obvious absorbance at 340 nm, so the change in absorbance at 340 nm can be monitored to determine the enzyme activity.

[0071] Select 10 mutant strains with the largest change rate of absorbance for subsequent shake flask rescreening.

[0072] Example 3

[0073] Shake flask rescreening of mutant strains

[0074] Inoculate the 10 monoclonal colonies obtained from the primary screening in the centrifuge tubes in Example 2 into 10 ml of LB liquid medium, culture at 37 °C until the OD reaches 1.8, then transfer to 200 ml of LB liquid medium. When the OD grows between 0.7 and 0.9, add IPTG with a final concentration of 0.4 mM and induce and culture effectively at 25 °C for more than 20 h.

[0075] After centrifuging the fermentation broth at the end of induction at 3000 rpm for 20 min, the precipitated cells were collected, 100 ml of cell lysis buffer was added, and after thorough lysis for 2 h, centrifugation was performed at 7000 rpm for 30 min. The supernatant was mixed upside down with 20 ml of Ni affinity chromatography packing for 30 min, and the supernatant was discarded; then 100 ml of washing buffer was added respectively to wash away the impurity proteins, and the operation method was the same as before; finally, HK was eluted with 50 ml of elution buffer, and the supernatant was collected, which was the enzyme solution. The cell lysis buffer was 100 mM Tris-HCl, 1.5% SDS, 200 U / ml lysozyme, 0.5% Tween 80, pH 8.0; the washing buffer consisted of 100 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0; the elution buffer consisted of 100 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, pH 8.0.

[0076] For the collected enzyme solution, the protein concentration was measured using a total protein detection kit, and according to the protein concentration of the enzyme solution, the protein was diluted to 0.05 g / L using the elution buffer. The absorbance change rate was measured using the enzyme activity measurement method in Example 2, and according to the magnitude of the absorbance change rate, a mutant strain with the highest catalytic activity was selected.

[0077] Example 4

[0078] Determination of mutant enzyme activity and thermal stability

[0079] The mutant strain with the highest catalytic activity obtained in Example 3 was inoculated into 10 ml of LB liquid medium, cultured at 37 °C until the OD reached 1.5, transferred to 2000 ml of 2YT liquid medium, cultured at 37 °C until the OD reached 1, 0.5 mM IPTG was added, and induction was carried out at 22 °C for more than 24 h.

[0080] The fermentation broth was centrifuged at 3000 rpm for 30 min to collect the cells, 10 times the volume of the disruption solution was added, ultrasonic disruption was carried out for 20 min, and after centrifugation at 8000 rpm for 60 min, the supernatant was taken. The supernatant was purified using Ni column affinity chromatography. First, the supernatant was passed through the Ni packing, and then the impurity proteins were washed away with the washing buffer, and the target protein was eluted with the elution buffer. The disruption solution consisted of: 50 mM Tris-HCl, 300 mM NaCl, 5 mM imidazole, pH 8.0; the washing buffer consisted of 50 mM Tris-HCl, 300 mM NaCl, 50 mM imidazole, pH 8.0; the elution buffer consisted of 50 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, pH 8.0.

[0081] The recovered enzyme solution was used to determine the protein concentration by the method in Example 3. The protein solution was diluted into a series of gradients with an enzyme activity dilution solution, and its specific enzyme activity was determined. The enzyme activity dilution solution was 100 mM Tris-HCl, pH 7.5, 1 g / L BSA.

[0082] The method for determining enzyme activity was as follows: 10 μl of the protein solution was added to 1 ml of the activity assay solution, and the reaction was carried out for 5 min. The change rate of absorbance at 340 nm in one minute between the 2nd minute and the 3rd minute was recorded. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the reaction of glucose and adenosine triphosphate to generate 1 μM of glucose-6-phosphate in 1 min.

[0083] The formula for calculating enzyme activity was:

[0084] Enzyme activity (U / ml) = ( ODmin × 101 × df) / 6.22

[0085] Where: ODmin: The change rate of absorbance in 1 min; 101: The dilution factor of the activity assay; df: The dilution factor of the protein solution; 6.22: The extinction coefficient of NADH.

[0086] The specific enzyme activity of the mutant strain was obtained by dividing the measured enzyme activity by the measured protein concentration.

[0087] According to the above protocol, the specific enzyme activity of the hexokinase mutant strain was determined to be 630 U / mg.

[0088] The HK enzyme solution was placed in a water bath at temperatures such as 30, 35, 40, 45, 50, 55, 60 °C for heat treatment for 30 min, and the enzyme activity was measured in the same way and compared with the untreated enzyme activity, so as to obtain the enzyme activity retention rate at different temperatures.

[0089] The enzyme activity retention rate of hexokinase at different temperatures is shown in Figure 1 . From Figure 1 it can be seen that after the enzyme was treated at 55 °C for 30 min, the enzyme activity retention rate was 62.1%.

[0090] Example 5

[0091] Performance verification of hexokinase in a glucose detection kit

[0092] The hexokinase enzyme solution prepared in Example 4 was added to the glucose detection kit, and the reagent was placed in a water bath at 37 °C for thermal storage for 11 days. Under normal calibration conditions, key indicators such as the accuracy, precision, and linear relationship of the analysis kit were measured. The composition of kit R1 is: 50 mM Tris-HCl, 2 KU / L hexokinase, 2 KU / L glucose-6-phosphate dehydrogenase, pH 8.0; the composition of R2 is: 50 mM Tris-HCl, 5 mM adenosine triphosphate, 3 mM NAD + , pH 9.0.

[0093] The accuracy determination of the kit was carried out under the normal calibration of the kit. The assigned quality control was measured, and the measured value was compared with the quality control. A deviation within ±5% was considered qualified for accuracy. The precision determination was carried out under the normal calibration of the kit. The high-value sample and the low-value sample were continuously measured 20 times respectively. After statistical analysis, the CV was calculated. A CV ≤ 2% was considered qualified for precision. The linear determination was carried out under the normal calibration of the kit. 40 mM linear high-value and normal saline were mixed at ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10. The measured value was subjected to regression analysis with the theoretical value, and R 2 ≥ 0.995 was considered qualified. The accuracy and precision determination results of hexokinase applied to the glucose detection kit are shown in Table 1. The linear relationship of HK applied to the glucose detection kit before and after thermal storage is shown in Figure 2 . It can be seen from Table 1 that before thermal storage, the low-value quality control and the high-value quality control deviated by -3.20% and 0.22% respectively, and after thermal storage, the low-value quality control and the high-value quality control deviated by -2.72% and 0.49% respectively, and the accuracy was qualified; before thermal storage, the CV of the low-value sample and the high-value sample were 1.30% and 1.89% respectively, and after thermal storage, the CV of the low-value sample and the high-value sample were 1.84% and 1.09% respectively, and the precision was qualified. It can be seen from Figure 2 that the linear regression equations before and after thermal storage are Y = 1.002X + 0.0095, R 2 = 0.9996; Y = 1.0034X + 0.00973, R 2 = 0.9999. The linear relationship is good.

[0094] Table 1: Accuracy and precision determination results of hexokinase applied to the glucose detection kit

[0095]

[0096] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be subject to the scope defined by the claims.

Claims

1. A hexokinase, characterized in that The amino acid sequence of the hexokinase is shown in SEQ ID NO.1; The hexokinase is obtained by mutation of the hexokinase gene of wild-type Saccharomyces cerevisiae; the gene mutation is that the amino acid sequence of the hexokinase of the wild-type Saccharomyces cerevisiae is modified by substitution of position 52 to E52V, and position 254 to S254P.

2. An application of hexokinase, characterized in that: The hexokinase according to claim 1 is used in a glucose detection kit.

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