Hexokinase mutant with high thermal stability and preparation and application thereof

Abstract

The present application relates to the field of biotechnology, in particular to a high-thermal-stability hexokinase mutant and preparation and application thereof, the hexokinase mutant is formed by amino acid mutations in the hexokinase shown in SEQ ID NO. 1, the amino acid mutations are selected from one or more of the following: 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 or V481I. The present application realizes the stability improvement (improvement of nearly 40%) of HXK hexokinase by expressing the mutant in E. coli, and the expression product can be used for the development and application of glucose detection related kits after purification.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to thermostable hexokinase mutants and their preparation and application. Background Technology

[0002] With the continuous improvement of living standards, the number of people with diabetes is surging, placing increasingly higher demands on the daily monitoring and early diagnosis of diabetes. Blood glucose levels are usually reflected by quantitative detection of glucose in serum or plasma, but in vitro detection requires a reaction catalyzed by hexokinase to achieve accurate quantification.

[0003] Hexokinase (HXK) is ubiquitous in organisms and a key enzyme in energy metabolism. It is a transferase with strong affinity, specifically catalyzing the reaction of hexoses with ATP to produce glucose-6-phosphate and ADP, thereby achieving energy transfer. This property can be utilized in the development of glucose detection kits. Most commercially available hexokinases are derived from natural extraction or recombinant expression in prokaryotes and eukaryotes. Natural extraction results in low yields and high purification difficulties, hindering industrial application. Recombinant expression allows for large-scale production, but the enzyme's stability remains a significant factor limiting its industrial application. Some manufacturers optimize kits by adding large amounts of surfactants to slow enzyme activity decline, but this significantly increases reagent costs and does not fundamentally solve the stability problem. Therefore, obtaining hexokinases with improved stability for large-scale reagent applications holds great promise. Summary of the Invention

[0004] To address the issues of low extraction yield, high purification difficulty, challenges in industrial application, high component content, and poor enzyme stability associated with hexokinase, this invention analyzes the function, protein structure, and key sites of wild-type hexokinase from Saccharomyces cerevisiae and designs numerous mutations to improve stability, providing a hexokinase mutation scheme with enhanced thermostability.

[0005] Specifically as follows:

[0006] A thermostable hexokinase mutant, wherein the hexokinase mutant is formed by an amino acid mutation in the hexokinase shown in SEQ ID NO.1, and 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 is mutated to aspartic acid H29D, asparagine H29N or glutamine H29Q;

[0008] (2) The 46th lysine residue is mutated to arginine K46R or proline K46P;

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

[0010] (4) The alanine at position 161 is mutated to serine A161S, threonine A161T or cysteine ​​A161C.

[0011] (5) The serine at position 255 is mutated to arginine S255R, histidine S255H or proline S255P.

[0012] (6) The lysine at position 284 is mutated to arginine K284R or histidine K284H;

[0013] (7) The 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 "thermally stable hexokinase mutant" mentioned in this invention refers to a mutated hexokinase that exhibits higher enzyme activity after heat treatment compared to wild-type hexokinase.

[0017] It should be noted that the above "the amino acid mutation is selected from one or more of the amino acid mutations (1)-(9)" refers to any 1 mutation, any 2 mutations, any 3 mutations, any 4 mutations, any 5 mutations, any 6 mutations, any 7 mutations, any 8 mutations, and the above 9 mutations among the amino acid mutations (1)-(9).

[0018] The nucleotide sequence of the hexokinase described in this invention is shown in SEQ ID NO.2. In the amino acid mutation (1), H29D is the 85th nucleotide where the C base is mutated to the G base, H29N is the 85th nucleotide where the C base is mutated to the A base, and H29Q is the 87th nucleotide where the C base is mutated to the A base.

[0019] In the amino acid mutation (2), K46R is the 137th nucleotide where the A base is mutated to the G base, and K46P is the 136th nucleotide where the A base is mutated to the C base and the 137th nucleotide is mutated to the C base.

[0020] In the amino acid mutation (3), E53A is the 158th nucleotide where the A base is mutated to the C base, E53G is the 158th nucleotide where the A base is mutated to the G base, E53L is the 157th nucleotide where the G base is mutated to the C base and the 158th nucleotide is changed from the A base, and E53V is the 158th nucleotide where the A base is mutated to the T base;

[0021] In the amino acid mutation (4), A161S is the 481st nucleotide where the G base is mutated to the T base, A161T is the 481st nucleotide where the G base is mutated to the A base, and A161C is the 481st nucleotide where the G base is mutated to the T base, the 482nd nucleotide is mutated to the G base, and the 483rd nucleotide is mutated to the C base.

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

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

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

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

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

[0027] In another aspect, the present invention discloses a reagent or kit containing the hexokinase mutant described above.

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

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

[0030] C1) encodes the nucleic acid molecule of 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) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3);

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

[0035] In the above-mentioned biological materials, the vector may be a plasmid, a granule, a bacteriophage, or a viral vector. Preferably, the recombinant vector is selected from pET28a, pET32a, or pBAD.

[0036] In the above-mentioned biological materials, the microorganisms may be yeast, bacteria, algae or fungi, and preferably, the microorganisms are Escherichia coli.

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

[0038] In another aspect, the present invention discloses a method for preparing the hexokinase mutant, comprising introducing the coding gene of the hexokinase mutant into a biological cell to express the coding gene of the hexokinase mutant, thereby obtaining the hexokinase mutant.

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

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

[0041] E2) Application of any of the biomaterials described above in the preparation of hexokinase mutants;

[0042] E3) Application of the hexokinase mutant in the preparation of glucose detection kits;

[0043] E4) The application of any of the biomaterials described above in the preparation of glucose detection products;

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

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

[0046] Beneficial effects

[0047] This invention provides 9 mutation sites and 28 unit site mutation methods. Mutating the 9 sites using the 28 unit site mutation methods can improve the thermal stability relative to the wild type of hexokinase. The 9 sites can be mutated individually or multiple sites can be mutated simultaneously. Mutating two or more sites simultaneously can further improve the thermal stability compared to a single mutation.

[0048] Each mutation site in this invention has more than two mutation modes, providing more combinations for the simultaneous selection of multiple mutation sites. This invention not only provides a reference for the research of hexokinase, but also provides more possibilities for its further application due to the stable performance of hexokinase mutants. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions in the embodiments of this disclosure will be clearly and completely described below. Where specific conditions are not specified in the examples or embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified 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 commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0051] Unless otherwise specified, the practice of this disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Methods in Enzymology* (Academic Press, Inc.); *Current Protocols in Molecular Biology* (edited by F.M. Ausubel et al., 1987); and *PCR: The Polymerase Chain Reaction* (edited by Mullis et al., 1994), each of which is expressly incorporated herein by reference.

[0052] Example 1: Obtaining the mutant protein

[0053] 1. Construction of the expression carrier

[0054] Based on the gene derived from *Saccharomyces cerevisiae* (GenBank Accession: NP_011261.1), and following the codon optimization principles of *E. coli*, corresponding nucleotide sequences were designed and synthesized by Shanghai Jierui Biotechnology Co., Ltd., and constructed into the pET28a expression vector. A total of 29 vectors were obtained (28 mutant genes and wild-type genes). The constructed expression vectors are pET28a-H29D, pET28a-H29N, pET28a-H29Q, pET28a-K46R, pET28a-K46P, pET28a-E53A, pET28a-E53G, pET28a-E53L, pET28a-E53V, and 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, induction of expression, and purification of recombinant bacteria

[0056] The above 29 vectors were transformed into Escherichia coli host BL21(DE3). The transformants were inoculated into LB medium and incubated overnight at 37°C. Then, they were transferred at a ratio of 1 / 100 to fresh LB medium and incubated at 37°C until OD (October Expiratory Time). 600 When the concentration of the bacterial cell was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM to induce expression. After 5 hours of induction, the bacterial cells were collected. After homogenization, nickel affinity chromatography was performed to obtain wild-type HXK cells and single mutants containing the His tag, which were used for further evaluation.

[0057] 3. HXK activity testing method

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

[0059] (2) Enzyme activity unit U: Under the conditions of 30℃ and pH 8.0, with glucose as substrate, the amount of hexokinase required to produce 1 μmol NADH per minute after 5 to 6 minutes of reaction is defined as the enzyme activity of one unit of hexokinase.

[0060] (3) Main reagents

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

[0062] b. Glucose solution: Prepare a 0.67M glucose solution using solution a above. It needs to be equilibrated to room temperature before use.

[0063] c. ATP solution: Prepare a 16.5 mM ATP solution using solution a above, and use immediately after preparation;

[0064] d.NAD + Solution: Prepare 6.8 mM NAD using solution a above. + The solution should be prepared and used immediately.

[0065] eG-6-PDH solution: Prepare a 300 U / mL G-6-PDH solution using the above solution a. Place on ice after preparation and use immediately.

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

[0067] g. HXK assay working solution: by volume ratio a: glucose solution: ATP solution: NAD + The solution is prepared by mixing G-6-PDH solution in a ratio of 230:50:10:10:1 and should be used immediately after preparation.

[0068] (4) Sample determination

[0069] Add 210.7 μL of HXK activity assay working solution to a 96-well UV detection plate using a multi-channel pipette, and preheat at 30°C for 5 min at 600 rpm in an insulated shaker. Transfer the diluted enzyme solution to an 8-tube PCR unit, and add 7 μL of the enzyme solution to the preheated working solution using a 10 μL multi-channel pipette. Stir clockwise 10 times and immediately place the tube in a microplate reader for measurement. Measure continuously for 6 min using the rate mode, measuring OD every 1 minute. 340nm The absorbance value at that location.

[0070] (5) Vitality calculation formula:

[0071] Activity concentration U / mL=5×ΔA340nm ×Activeness Dilution Factor

[0072]

[0073] ΔA 340nm =ΔOD 5min -ΔOD 4min

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

[0075] Wild-type protein and 28 mutant proteins were diluted to 1.0 mg / mL using HXK enzyme dilution buffer and aliquoted into 1.5 mL centrifuge tubes (500 μL per tube). Four tubes were aliquoted for each protein. Two tubes were heat-treated in a 52°C water bath for 30 min, and the other two tubes were kept on ice until 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 activity of wild-type hexokinase and 28 single mutant proteins

[0078]

[0079]

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

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

[0082] In this embodiment, to investigate the combined effect of multiple mutations on the stability of hexokinase, multiple mutation sites were constructed based on the test results of a single mutation in Example 1. Following the E. coli codon optimization principle, corresponding nucleotide sequences were designed, synthesized by Shanghai Jierui Biotechnology Co., Ltd., and constructed into the pET28a expression vector, resulting in a total of 8 vectors. The constructed expression vectors are as follows:

[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, induction of expression, and purification of recombinant bacteria

[0092] The above eight vectors were transformed into Escherichia coli host BL21(DE3). The transformants were inoculated into LB medium and incubated overnight at 37°C. Then, they were transferred at a ratio of 1 / 100 to fresh LB medium and incubated at 37°C until OD (October Expiratory Time). 600 When the concentration of the bacterial cell was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM to induce expression. After 5 hours of induction, the bacterial cells were collected. After homogenization, nickel affinity chromatography was performed to obtain multiple mutants containing the His tag for subsequent evaluation.

[0093] 2. Stability test of pure enzyme solution

[0094] Wild-type protein and eight mutant proteins were diluted to 1.0 mg / mL using HXK enzyme dilution buffer and aliquoted into 1.5 mL centrifuge tubes (500 μL per tube). Four tubes were aliquoted for each protein. Two tubes were heat-treated in a 52°C water bath for 30 min, and the other two tubes were kept on ice until 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 2.

[0095] Table 2 Residual activity of wild-type hexokinase and 8 multi-mutant proteins

[0096] 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] Based on the validation results of single mutants, the optimal mutations at a single point were combined to construct the above 8 mutants. The results show that simultaneous mutation at multiple mutation points (more than 2) is more effective than a single mutation point, and the mutation sites (1) to (9) listed in this invention can be randomly combined. Among the current combined mutations, the mutation combinations of V481I-G408N-A161S-K46P-S255P, V481I-G408N-A161S-K46P-S255P-H29N, and V481I-G408N-A161S-K46P-S255P-I403L have better effects, with a stability improvement of over 40% compared to the wild type.

[0098] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A thermostable hexokinase mutant, characterized in that, The hexokinase mutant is formed by amino acid mutations in the hexokinase shown in SEQ ID NO.1, wherein the amino acid mutations are V481I, G408N, A161S, K46P, S255P, and H29N.

2. The hexokinase mutant of claim 1, wherein the nucleotide sequence of the hexokinase is shown in SEQ ID NO.2, characterized in that, The V481I is a nucleotide where the 1441st nucleotide is mutated from a G base to an A base; The G408N is a mutation where the nucleotide at position 1222 is changed from a G base to an A base and the nucleotide at position 1223 is changed from a G base to an A base. The A161S is a nucleotide where the 481st nucleotide is mutated from a G base to a T base; The K46P is a mutation where the nucleotide at position 136 is changed from an A base to a C base and the nucleotide at position 137 is changed from an A base to a C base. The S255P is a nucleotide at position 763 where a T base is mutated to a C base. The H29N is a nucleotide at position 85 where a C base is mutated to an A base.

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

4. A biological material relating to the hexokinase mutant of claim 1 or 2, wherein the biological material is any one of C1) to C5) below: C1) encodes the nucleic acid molecule of 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) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3); C5) A transgenic cell line containing the nucleic acid molecule described in C1) or a transgenic cell line containing the expression cassette described in C2), wherein the transgenic cell line does not include propagation material.

5. The biomaterial as described in claim 4, characterized in that, The vector is a plasmid or a viral vector.

6. The biomaterial as described in claim 5, characterized in that, The recombinant vector is selected from pET28a, pET32a, and pBAD.

7. The biomaterial as described in claim 4, characterized in that, The microorganism in question is Escherichia coli.

8. The method for preparing the hexokinase mutant as described in claim 1 or 2, characterized in that, The preparation method includes 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.

9. The following application, characterized in that, E1) The application of the hexokinase mutant as described in claim 1 or 2 in the preparation of biocatalysts; E2) The use of the biomaterials described in any one of claims 4-7 in the preparation of hexokinase mutants; E3) The use of the hexokinase mutant as described in claim 1 or 2 in the preparation of a glucose detection kit; E4) The use of the biomaterials described in any one of claims 4-7 in the preparation of glucose detection products.

10. The application as described in claim 9, characterized in that, The biocatalyst catalyzes the reaction of hexoses and ATP to produce glucose-6-phosphate and ADP.

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