Hexokinase mutant with high thermal stability as well as preparation and application of hexokinase mutant

Abstract

The invention relates to the technical field of biology, in particular to a hexokinase mutant with high thermal stability and preparation and application of the hexokinase mutant, the hexokinase mutant is formed by amino acid mutation in hexokinase as shown in SEQ ID NO.1, and the amino acid mutation is selected from one or more of H29D, H29N, H29Q, K46R, K46P, E53A, E53G, E53L, E53V, A161S, A161T, A161C, S255R, S255H, S255P, K284R, K284H, I403A, I403G, I403L, I403V, G408D, G408N, G408E, V481A, V481G, V481L The mutant is expressed in escherichia coli, so that the stability of HXK hexokinase is improved by about 40%, and the expression product can be used for development and application of related kits for glucose detection after being purified.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a hexokinase mutant with high thermal stability and a preparation and application thereof. Background Art

[0002] With the continuous improvement of living standards, the number of people with diabetes has increased sharply, and higher and higher requirements have been put forward for the daily monitoring and early diagnosis of diabetes. Blood sugar levels are usually reflected by quantitative detection of glucose in serum or plasma, and the realization of in vitro detection requires a reaction catalyzed by hexokinase to achieve accurate quantification.

[0003] Hexokinase (HXK) is ubiquitous in organisms and is a key enzyme in energy metabolism. It is a transferase with strong affinity, which specifically catalyzes the reaction of hexose and ATP to produce glucose-6-phosphate and ADP, thereby realizing energy transfer. This characteristic can be used to develop glucose detection kits. Most of the hexokinases used on the market are derived from natural extraction or recombinant expression of prokaryotes and eukaryotes. Natural extraction has low yield, high purification difficulty, and difficult industrial application. Recombinant expression can achieve large-scale preparation, but the stability of the enzyme itself has become an important factor restricting its industrial application. Some manufacturers optimize the kit and add a large amount of surfactants to slow down the attenuation of enzyme activity, but the added raw materials greatly increase the reagent cost and cannot fundamentally solve the problem of stability. Therefore, it is very promising to obtain hexokinase with improved stability to achieve large-scale application on the reagent side. Summary of the invention

[0004] In order to solve the problems of low extraction yield, high purification difficulty, difficulty in industrial application, high component and poor enzyme stability of the above-mentioned hexokinase, the present invention analyzes the function, protein structure and key sites of the sequence of wild-type hexokinase of Saccharomyces cerevisiae, designs a large number of mutations for stability modification, and provides a hexokinase mutation scheme with improved thermal stability.

[0005] The details are as follows:

[0006] A hexokinase mutant with high thermal stability, wherein the hexokinase mutant is formed by an amino acid mutation in the hexokinase shown in SEQ ID NO.1, wherein the amino acid mutation is selected from one or more of the following amino acid mutations (1)-(9):

[0007] (1) The histidine at position 29 was mutated to aspartic acid H29D, asparagine H29N, or glutamine H29Q;

[0008] (2) Lysine 46 is mutated to arginine K46R or proline K46P;

[0009] (3) glutamic acid at position 53 is mutated to alanine E53A, glycine E53G, leucine E53L, or valine E53V;

[0010] (4) alanine 161 mutated to serine A161S, threonine A161T, or cysteine ​​A161C;

[0011] (5) Serine 255 was mutated to arginine S255R, histidine S255H, or proline S255P;

[0012] (6) Lysine 284 was mutated to arginine K284R or histidine K284H;

[0013] (7) Isoleucine at position 403 is mutated to alanine I403A, glycine I403G, leucine I403L, or valine I403V;

[0014] (8) Glycine at position 408 is mutated to aspartic acid G408D, asparagine G408N, or glutamic acid G408E;

[0015] (9) The valine at position 481 is mutated to alanine V481A, glycine V481G, leucine V481L or isoleucine V481I.

[0016] The "hexokinase mutant with high thermal stability" described in the present invention refers to a mutated hexokinase having higher enzyme activity after heat treatment than wild hexokinase.

[0017] It should be noted that the above “the amino acid mutation is selected from one or more of amino acid mutations (1)-(9)” refers to any one mutation, any two mutations, any three mutations, any four mutations, any five mutations, any six mutations, any seven mutations, any eight mutations and the above nine mutations among amino acid mutations (1)-(9).

[0018] The nucleotide sequence of the hexokinase of the present invention is shown in SEQ ID NO.2. In the amino acid mutation (1), the H29D is a mutation of the nucleotide at position 85 from C to G, the H29N is a mutation of the nucleotide at position 85 from C to A, and the H29Q is a mutation of the nucleotide at position 87 from C to A.

[0019] In the amino acid mutation (2), the K46R is a mutation of the nucleotide at position 137 from an A base to a G base, and the K46P is a mutation of the nucleotide at position 136 from an A base to a C base and a mutation of the nucleotide at position 137 from an A base to a C base;

[0020] In the amino acid mutation (3), the E53A is a mutation of the nucleotide at position 158 from an A base to a C base, the E53G is a mutation of the nucleotide at position 158 from an A base to a G base, the E53L is a mutation of the nucleotide at position 157 from a G base to a C base and the nucleotide at position 158 from an A base, and the E53V is a mutation of the nucleotide at position 158 from an A base to a T base;

[0021] In the amino acid mutation (4), the A161S is a mutation of the 481st nucleotide from a G base to a T base, the A161T is a mutation of the 481st nucleotide from a G base to an A base, the A161C is a mutation of the 481st nucleotide from a G base to a T base, the 482nd nucleotide from a C base to a G base, and the 483rd nucleotide from an A base to a C base;

[0022] In the amino acid mutation (5), the S255R is a mutation of the nucleotide at position 763 from a T base to a C base and the nucleotide at position 764 from a C base to a G base, the S255H is a mutation of the nucleotide at position 763 from a T base to a C base and the nucleotide at position 764 from a C base to an A base, and the S255P is a mutation of the nucleotide at position 763 from a T base to a C base;

[0023] In the amino acid mutation (6), the K284R is a mutation of the nucleotide at position 850 from an A base to a G base, and the K284H is a mutation of the nucleotide at position 850 from an A base to a C base and a mutation of the nucleotide at position 852 from an A base to a C base;

[0024] In the amino acid mutation (7), the I403A is a mutation of the nucleotide at position 1207 from an A base to a G base and the nucleotide at position 1208 from a T base to a C base, the I403G is a mutation of the nucleotide at position 1207 from an A base to a G base and the nucleotide at position 1208 from a T base to a G base, the I403L is a mutation of the nucleotide at position 1207 from an A base to a C base, and the I403V is a mutation of the nucleotide at position 1207 from an A base to a G base;

[0025] In the amino acid mutation (8), the G408D is a mutation of the nucleotide at position 1223 from a G base to an A base, the G408N is a mutation of the nucleotide at position 1222 from a G base to an A base and the nucleotide at position 1223 from a G base to an A base, and the G408E is a mutation of the nucleotide at position 1223 from a G base to an A base and the nucleotide at position 1224 from a T base to an A base;

[0026] Or in the amino acid mutation (9), the V481A is a mutation of the nucleotide at position 1442 from a T base to a C base, the V481G is a mutation of the nucleotide at position 1442 from a T base to a G base, the V481L is a mutation of the nucleotide at position 1441 from a G base to a C base, and the V481I is a mutation of the nucleotide at position 1441 from a G base to an A base.

[0027] Another aspect of the present invention discloses a reagent or a kit, wherein the reagent or the kit contains the hexokinase mutant.

[0028] In another aspect, the present invention discloses biological materials related to the hexokinase mutant.

[0029] The biological material is any one of the following C1) to C5):

[0030] C1) a nucleic acid molecule encoding the hexokinase mutant;

[0031] C2) an expression cassette containing the nucleic acid molecule described in C1);

[0032] C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0033] C4) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3);

[0034] C5) A transgenic cell line comprising the nucleic acid molecule described in C1), or a transgenic cell line comprising the expression cassette described in C2), wherein the transgenic cell line does not comprise propagation material.

[0035] In the above biological materials, the vector may be a plasmid, a cosmid, a phage or a viral vector. Preferably, the vector in the recombinant vector is selected from pET28a, pET32a and pBAD.

[0036] In the above biological material, the microorganism may be yeast, bacteria, algae or fungi. Preferably, the microorganism is Escherichia coli.

[0037] Preferably, the microorganism is Escherichia coli BL21 (DE3).

[0038] Another aspect of the present invention discloses a method for preparing the hexokinase mutant, comprising introducing the coding gene of the hexokinase mutant into biological cells to express the coding gene of the hexokinase mutant to obtain the hexokinase mutant.

[0039] In another aspect, the present invention discloses any of the following applications:

[0040] E1) Use of the hexokinase mutant as a biocatalyst;

[0041] E2) Use of any of the above biological materials in the preparation of hexokinase mutants;

[0042] E3) Use of the hexokinase mutant in preparing a glucose detection kit;

[0043] E4) Use of any of the above biological materials in the preparation of glucose detection products;

[0044] E5) Use of the hexokinase mutant described in diabetes detection products.

[0045] Preferably, the biocatalyst catalyzes the reaction of hexose and ATP to produce glucose-6-phosphate and ADP.

[0046] Beneficial Effects

[0047] The present invention provides 9 mutation sites and 28 single-point mutation methods. The 9 sites are mutated by using the 28 single-point mutation methods, which can all improve the thermal stability relative to the wild-type hexokinase. The 9 sites can be mutated individually or multiple sites can be mutated simultaneously. The simultaneous mutation of more than two mutation sites can further improve the thermal stability relative to a single mutation.

[0048] Each mutation site of the present invention has more than two mutation modes, which provides more combination modes for the simultaneous selection of multiple mutation sites. The present invention not only provides a reference for the study of hexokinase, but also the stable performance of the hexokinase mutant provides more possibilities for its further application. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the embodiments of the present disclosure clearer, the technical scheme in the embodiments of the present disclosure will be described clearly and completely below. If no specific conditions are specified in the examples or embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0051] Unless otherwise indicated, the present disclosure will be practiced using the conventional techniques of cell biology, molecular biology (comprising recombinant technology), microbiology, biochemistry and immunology, which are within the capabilities of those skilled in the art. This technology is fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, 1984); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (FM Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al., 1994), each of which is expressly incorporated herein by reference.

[0052] Example 1 Obtaining mutant protein

[0053] 1. Construction of expression vector

[0054] According to the gene from Saccharomyces cerevisiae (GenBank Accession: NP_011261.1), the corresponding nucleotide sequence was designed according to the codon optimization principle of Escherichia coli, and it was synthesized by Shanghai Jierui Biological Company and constructed into the pET28a expression vector. A total of 29 vectors (28 mutant genes and wild-type genes) were obtained. The constructed expression vectors were pET28a-H29D, pET28a-H29N, pET28a-H29Q, pET28a-K46R, pET28a-K46P, pET28a-E53A, pET28a-E53G, pET28a-E53L, pET28a-E53V, pET28a-A 161S, pET28a-A161T, pET28a-A161C, pET28a-S255R, pET28a-S255H, pET28a-S255P, pET28a-K284R, pET28a-K284H, pET28a-I403A, pET28a-I403G, pET 28a-I403L, pET28a-I403V, ​​pET28a-G408D, pET28a-G408N, pET28a-G408E, pET28a-V481A, pET28a-V481G, pET28a-V481L, pET28a-V481I, pET28a-wild type.

[0055] 2. Construction, induced expression and purification of recombinant bacteria

[0056] The above 29 vectors were transformed into E. coli host BL21 (DE3), and the transformants were inoculated into LB medium and cultured at 37°C overnight. Then, they were transferred to fresh LB medium at a rate of 1 / 100 and cultured at 37°C until OD 600 =0.6-0.8, add IPTG at a final concentration of 0.5 mM to induce expression, collect the cells after 5 h of induction, and perform nickel affinity chromatography after being broken by a homogenizer to obtain wild-type HXK and single mutants containing His tags for subsequent evaluation.

[0057] 3. HXK activity test method

[0058] (1) Detection principle: Hexokinase catalyzes the reaction of glucose and ATP to generate glucose-6-phosphate, which then reacts with NAD + NADH is generated under the catalysis of G-6-PDH, and NADH has a characteristic absorption peak at 340nm. The activity of hexokinase can be determined by measuring the change in the absorbance value of the reaction at 340nm.

[0059] (2) Enzyme activity unit U: The amount of hexokinase required to produce 1 μmol NADH per minute after 5 to 6 minutes of reaction at 30°C and pH 8.0 with glucose as substrate is defined as one unit of hexokinase activity;

[0060] (3) Main reagents

[0061] a. Tris-HCl solution, pH = 8.0: Prepare 50 mM Tris-HCl solution, adjust pH to 8.0 with HCl, add MgCl2 to a final concentration of 13.3 mM, and store at 2-8°C;

[0062] b. Glucose solution: Use solution a to prepare 0.67M glucose solution, and equilibrate to room temperature before use;

[0063] c. ATP solution: Use the above solution a to prepare 16.5 mM ATP solution, and use it immediately after preparation;

[0064] d.NAD + Solution: Prepare 6.8mM NAD using solution a above + Solution, ready to use;

[0065] eG-6-PDH solution: Use the above solution a to prepare 300U / mL G-6-PDH solution, place it on ice after preparation, and use it immediately after preparation;

[0066] f. HXK enzyme dilution solution: Add BSA to the above solution a to a final concentration of 0.1% to obtain HXK enzyme dilution solution, and store at 2-8°C;

[0067] g. HXK activity test working solution: by volume ratio a solution: glucose solution: ATP solution: NAD + Solution: G-6-PDH solution = 230:50:10:10:1, prepared and used immediately.

[0068] (4) Sample measurement

[0069] Use a dispenser to draw 210.7 μL of HXK activity test working solution into a 96-well UV detection plate, and place it in a constant temperature oscillator at 600 rpm and preheat at 30°C for 5 minutes; transfer the diluted enzyme solution to 8-row PCR tubes, use a 10 μL dispenser to draw 7 μL of enzyme solution into the preheated working solution, stir clockwise for 10 circles, and immediately place it in an ELISA reader for measurement; use the rate mode to measure continuously for 6 minutes on the ELISA reader, and measure the OD every 1 minute. 340nm The absorbance value at .

[0070] (5) Vitality calculation formula:

[0071] Activity concentration U / mL = 5 × ΔA340nm × Activity test dilution multiple

[0072]

[0073] ΔA 340nm = ΔOD 5min -ΔOD 4min

[0074] 4. Stability test method of HXK pure enzyme solution

[0075] The wild-type protein and 28 mutant proteins were diluted to 1.0 mg / mL with HXK enzyme diluent, and dispensed into 1.5 mL centrifuge tubes, 500 μL per tube, and each protein was dispensed into 4 tubes, 2 tubes were placed in a 52°C water bath for heat treatment for 30 min, and 2 tubes were placed on ice for standby use. After all treatments were completed, the activity concentration of all samples was measured, and the residual activity percentage was calculated. The results are shown in Table 1.

[0076]

[0077] Table 1 Residual activities of wild-type hexokinase and 28 single mutant proteins

[0078]

[0079]

[0080] As can be seen from Table 1, the stability of the 28 constructed single mutants was improved compared with the wild type.

[0081] Example 2 Combination mutants with improved stability of HXK hexokinase

[0082] In order to explore the effect of multiple mutations on the stability of hexokinase, multiple point mutations were constructed according to the test results of the single mutation in Example 1. The corresponding nucleotide sequences were designed according to the codon optimization principle of Escherichia coli, and were synthesized by Shanghai Jierui Biological Company and constructed into the pET28a expression vector. A total of 8 vectors were obtained, and the constructed expression vectors were:

[0083] ①pET28a-V481I-G408N

[0084] ②pET28a-V481I-G408N-A161S

[0085] ③pET28a-V481I-G408N-A161S-K46P

[0086] ④pET28a-V481I-G408N-A161S-E53L

[0087] ⑤pET28a-V481I-G408N-A161S-K46P-S255P

[0088] ⑥pET28a-V481I-G408N-A161S-K46P-K284H

[0089] ⑦pET28a-V481I-G408N-A161S-K46P-S255P-H29N

[0090] ⑧pET28a-V481I-G408N-A161S-K46P-S255P-I403L

[0091] 1. Construction, induced expression and purification of recombinant bacteria

[0092] The above eight vectors were transformed into E. coli host BL21 (DE3), and the transformants were inoculated into LB medium and cultured at 37°C overnight. Then, they were transferred to fresh LB medium at a rate of 1 / 100 and cultured at 37°C until OD 600 =0.6-0.8, add IPTG at a final concentration of 0.5 mM to induce expression, collect the cells after 5 hours of induction, and perform nickel affinity chromatography after being broken by a homogenizer to obtain multiple mutants containing His tags for subsequent evaluation.

[0093] 2. Pure enzyme solution stability test

[0094] The wild-type protein and the eight mutant proteins were diluted to 1.0 mg / mL with HXK enzyme diluent, and dispensed into 1.5 mL centrifuge tubes, 500 μL per tube, and each protein was dispensed into 4 tubes, 2 tubes were placed in a 52°C water bath for heat treatment for 30 min, and 2 tubes were placed on ice for standby use. After all treatments were completed, the activity concentrations of all samples were measured, and the residual activity percentages were calculated. The results are shown in Table 2.

[0095] Table 2 Residual activities of wild-type hexokinase and eight mutant proteins

[0096] protein Residual activity% wild type 49.52 V481I-G408N 85.04 V481I-G408N-A161S 81.56 V481I-G408N-A161S-K46P 88.66 V481I-G408N-A161S-E53L 78.65 V481I-G408N-A161S-K46P-S255P 90.05 V481I-G408N-A161S-K46P-K284H 81.05 V481I-G408N-A161S-K46P-S255P-H29N 92.56 V481I-G408N-A161S-K46P-S255P-I403L 89.47

[0097] According to the verification results of the single mutants, the optimal mutations of the single points were combined to construct the above 8 mutants. The results show that the simultaneous mutation of multiple mutation points (more than 2) is better than that of a single mutation point, and the mutation sites (1) to (9) listed in the present invention can be randomly combined. Among the current combined mutations, the mutation combination of V481I-G408N-A161S-K46P-S255P, V481I-G408N-A161S-K46P-S255P-H29N, and V481I-G408N-A161S-K46P-S255P-I403L has a better effect, and the stability improvement effect exceeds 40% compared with the wild type.

[0098] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A hexokinase mutant with high thermal stability, characterized in that The hexokinase mutant is formed by amino acid mutation in the hexokinase shown in SEQ ID NO.1, wherein the amino acid mutation is selected from one or more of the following amino acid mutations (1)-(9): (1) The histidine at position 29 was mutated to aspartic acid H29D, asparagine H29N, or glutamine H29Q; (2) Lysine 46 is mutated to arginine K46R or proline K46P; (3) glutamic acid at position 53 is mutated to alanine E53A, glycine E53G, leucine E53L, or valine E53V; (4) alanine 161 mutated to serine A161S, threonine A161T, or cysteine ​​A161C; (5) Serine 255 was mutated to arginine S255R, histidine S255H, or proline S255P; (6) Lysine 284 was mutated to arginine K284R or histidine K284H; (7) Isoleucine at position 403 is mutated to alanine I403A, glycine I403G, leucine I403L, or valine I403V; (8) Glycine at position 408 is mutated to aspartic acid G408D, asparagine G408N, or glutamic acid G408E; (9) The valine at position 481 is mutated to alanine V481A, glycine V481G, leucine V481L or isoleucine V481I.

2. The hexokinase mutant according to claim 1, wherein the nucleotide sequence of the hexokinase is as shown in SEQ ID NO.2, wherein: In the amino acid mutation (1), the H29D is a mutation of the nucleotide at position 85 from a C base to a G base, the H29N is a mutation of the nucleotide at position 85 from a C base to an A base, and the H29Q is a mutation of the nucleotide at position 87 from a C base to an A base; In the amino acid mutation (2), the K46R is a mutation of the nucleotide at position 137 from an A base to a G base, and the K46P is a mutation of the nucleotide at position 136 from an A base to a C base and a mutation of the nucleotide at position 137 from an A base to a C base; In the amino acid mutation (3), the E53A is a mutation of the nucleotide at position 158 from an A base to a C base, the E53G is a mutation of the nucleotide at position 158 from an A base to a G base, the E53L is a mutation of the nucleotide at position 157 from a G base to a C base and the nucleotide at position 158 from an A base, and the E53V is a mutation of the nucleotide at position 158 from an A base to a T base; In the amino acid mutation (4), the A161S is a mutation of the 481st nucleotide from a G base to a T base, the A161T is a mutation of the 481st nucleotide from a G base to an A base, the A161C is a mutation of the 481st nucleotide from a G base to a T base, the 482nd nucleotide from a C base to a G base, and the 483rd nucleotide from an A base to a C base; In the amino acid mutation (5), the S255R is a mutation of the nucleotide at position 763 from a T base to a C base and the nucleotide at position 764 from a C base to a G base, the S255H is a mutation of the nucleotide at position 763 from a T base to a C base and the nucleotide at position 764 from a C base to an A base, and the S255P is a mutation of the nucleotide at position 763 from a T base to a C base; In the amino acid mutation (6), the K284R is a mutation of the nucleotide at position 850 from an A base to a G base, and the K284H is a mutation of the nucleotide at position 850 from an A base to a C base and a mutation of the nucleotide at position 852 from an A base to a C base; In the amino acid mutation (7), the I403A is a mutation of the nucleotide at position 1207 from an A base to a G base and the nucleotide at position 1208 from a T base to a C base, the I403G is a mutation of the nucleotide at position 1207 from an A base to a G base and the nucleotide at position 1208 from a T base to a G base, the I403L is a mutation of the nucleotide at position 1207 from an A base to a C base, and the I403V is a mutation of the nucleotide at position 1207 from an A base to a G base; In the amino acid mutation (8), the G408D is a mutation of the nucleotide at position 1223 from a G base to an A base, the G408N is a mutation of the nucleotide at position 1222 from a G base to an A base and the nucleotide at position 1223 from a G base to an A base, and the G408E is a mutation of the nucleotide at position 1223 from a G base to an A base and the nucleotide at position 1224 from a T base to an A base; Or in the amino acid mutation (9), the V481A is a mutation of the nucleotide at position 1442 from a T base to a C base, the V481G is a mutation of the nucleotide at position 1442 from a T base to a G base, the V481L is a mutation of the nucleotide at position 1441 from a G base to a C base, and the V481I is a mutation of the nucleotide at position 1441 from a G base to an A base.

3. A reagent or a kit, characterized in that: The reagent or kit contains the hexokinase mutant as claimed in claim 1 or 2.

4. A biological material related to the hexokinase mutant of claim 1 or 2, The biological material is any one of the following C1) to C5): C1) a nucleic acid molecule encoding the hexokinase mutant; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3); C5) A transgenic cell line comprising the nucleic acid molecule described in C1), or a transgenic cell line comprising the expression cassette described in C2), wherein the transgenic cell line does not comprise propagation material.

5. The biomaterial according to claim 4, characterized in that The vector may be a plasmid, a cosmid, a phage or a viral vector. Preferably, the vector in the recombinant vector is selected from pET28a, pET32a and pBAD.

6. The biomaterial according to claim 4, characterized in that The microorganism is selected from yeast, bacteria, algae or fungi. Preferably, the microorganism is Escherichia coli.

7. The method for preparing a hexokinase mutant according to claim 1 or 2, characterized in that: The preparation method comprises the steps of introducing the coding gene of the hexokinase mutant into biological cells to express the coding gene of the hexokinase mutant, thereby obtaining the hexokinase mutant.

8. Any of the following applications, characterized in that: E1) Use of the hexokinase mutant as claimed in claim 1 or 2 as a biocatalyst; E2) Use of the biomaterial according to any one of claims 4 to 6 in the preparation of a hexokinase mutant; E3) Use of the hexokinase mutant as claimed in claim 1 or 2 in the preparation of a glucose detection kit; E4) Use of the biomaterial as claimed in any one of claims 4 to 6 in the preparation of a glucose detection product; E5) Use of the hexokinase mutant as described in claim 1 or 2 in a diabetes detection product.

9. The use according to claim 8, characterized in that The biocatalyst catalyzes the reaction of hexose and ATP to produce glucose-6-phosphate and ADP.

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