A glucose dehydrogenase mutant and application thereof

Abstract

The application provides a glucose dehydrogenase mutant and application thereof, the mutant is obtained by point mutation of glucose dehydrogenase, and the mutation sites are the 44th, 176th, 253rd and / or 354th from the N terminal; compared with the wild type, the NMN + Activity is significantly improved. The glucose dehydrogenase mutant is used for synthesizing mannitol, and the synthesis method is catalytic synthesis of mannitol under the joint action of mannitol dehydrogenase, artificial coenzyme and the glucose dehydrogenase mutant, and the regeneration of artificial coenzyme NMN (H) in the reaction is realized. Two modules containing the mutant: artificial coenzyme NMN-mannitol dehydrogenase-coenzyme regeneration enzyme glucose dehydrogenase mutant and artificial coenzyme NMN-myocardial xanthine-coenzyme regeneration enzyme glucose dehydrogenase mutant, through the glucose dehydrogenase mutant, NMN + Is reduced back to NMNH, so that the coenzyme can be recycled, and the consumption of the coenzyme is reduced.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme catalysis, and in particular to a glucose dehydrogenase mutant and its applications. Background Technology

[0002] Glucose dehydrogenase is a biocatalyst that catalyzes the oxidation of glucose, specifically breaking down glucose molecules into gluconic acid, accompanied by the generation of hydride ions. This enzyme also requires the oxidized coenzyme NAD+ to function. + or NADP + The involvement of the electron acceptor NAD + or NADP + It quickly combines with the generated hydrogen anions to produce reduced coenzymes NADH or NADPH.

[0003] Due to its substrate specificity and excellent catalytic efficiency in generating reducing cofactors, GDH has been widely used in glucose sensors or as a unique cofactor regenerator in biocatalysis. Oxidized coenzyme NAD is required during the synthesis of valuable chemicals. + or NADP + However, the high cost and poor thermal stability of natural coenzymes greatly limit their industrial application. Using low-cost, highly stable artificial coenzymes is one effective way to reduce the industrial application cost of NAD(P)-preferring oxidoreductases. Nicotinamide mononucleotide (NMN) + Due to its good stability, low price, and the fact that, as a truncated form of natural coenzyme, its redox potential is similar to that of natural coenzyme, it can directly replace NAD(P). + It is applied in redox systems. Therefore, developing efficient coenzyme regeneration systems and redox enzyme elements that match them has strong application value, and the construction of their recycling systems is also of great economic significance.

[0004] Mannitol is a widely used sugar alcohol with important applications in the pharmaceutical field. It is used not only as a diuretic to treat cerebral edema and lower intraocular pressure, but also frequently for the prevention and treatment of renal failure. Furthermore, mannitol is used as a sweetener in the food industry because of its low calorie content and lack of rapid spikes in blood sugar, making it suitable for diabetics. In the cosmetics industry, mannitol is added to skincare products for its moisturizing properties, helping to keep skin soft and supple. Therefore, from both a health and economic perspective, synthetic mannitol has significant value and broad market demand. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a glucose dehydrogenase mutant.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned glucose dehydrogenase mutant.

[0007] A glucose dehydrogenase mutant is obtained by point mutation of glucose dehydrogenase. The amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO.1, with mutation sites at positions 44, 176, 253, and / or 354 from the N-terminus.

[0008] The point mutation at position 44 starting from the N-terminus is E44H, the point mutation at position 176 is D176S, D176C, D176E, or D176Q, the point mutation at position 253 is A253V, and the point mutation at position 354 is K354R.

[0009] For amino acid substitutions, the following nomenclature is used: original amino acid (wild type) and the corresponding substitution amino acid at the same position. For example, histidine is used to replace the original glutamic acid at position 44, resulting in "E44H".

[0010] The above-mentioned glucose dehydrogenase mutant, wherein the glucose dehydrogenase is a glucose dehydrogenase derived from Sulfolobus solfatarcus.

[0011] Preferably, the above-mentioned glucose dehydrogenase mutant includes:

[0012] (I) The nucleic acid molecule encoding the glucose dehydrogenase mutant;

[0013] (II) An expression cassette, recombinant vector, or recombinant bacteria containing the nucleic acid molecule;

[0014] The nucleic acid molecule encoding the glucose dehydrogenase from Sulfolobus solfataricus (wild-type glucose dehydrogenase gene gdh) is the DNA molecule shown in SEQ ID NO.9.

[0015] The recombinant bacteria is Escherichia coli containing the nucleic acid molecule.

[0016] Preferably, the above-mentioned glucose dehydrogenase mutant has a mutation site at position 44 from the N-terminus: E44H, and its amino acid sequence is shown in SEQ ID NO.2; more preferably, its nucleotide sequence is shown in SEQ ID NO.10.

[0017] Preferably, the above-mentioned glucose dehydrogenase mutant has mutation sites at positions 44 and 176 from the N-terminus: E44H and D176S, and its amino acid sequence is shown in SEQ ID NO.3; more preferably, its nucleotide sequence is shown in SEQ ID NO.11.

[0018] Preferably, the above-mentioned glucose dehydrogenase mutant has mutation sites at positions 44 and 176 from the N-terminus: E44H and D176C, and its amino acid sequence is shown in SEQ ID NO.4; more preferably, its nucleotide sequence is shown in SEQ ID NO.12.

[0019] Preferably, the above-mentioned glucose dehydrogenase mutant has mutation sites at positions 44 and 176 from the N-terminus: E44H and D176E, and its amino acid sequence is shown in SEQ ID NO.5; more preferably, its nucleotide sequence is shown in SEQ ID NO.13.

[0020] Preferably, the above-mentioned glucose dehydrogenase mutant has mutation sites at positions 44 and 176 from the N-terminus: E44H and D176Q, and its amino acid sequence is shown in SEQ ID NO. 6 of the sequence listing; more preferably, its nucleotide sequence is shown in SEQ ID NO. 14 of the sequence listing.

[0021] Preferably, the above-mentioned glucose dehydrogenase mutant has mutation sites at positions 44, 176, and 253 from the N-terminus: E44H, D176S, and A253V, and its amino acid sequence is shown in SEQ ID NO.7 of the sequence listing; more preferably, its nucleotide sequence is shown in SEQ ID NO.15 of the sequence listing.

[0022] Preferably, the above-mentioned glucose dehydrogenase mutant has mutation sites at positions 44, 176, 253, and 354 from the N-terminus: E44H, D176S, A253V, and K354R, and its amino acid sequence is shown in SEQ ID NO. 8 of the sequence listing; more preferably, its nucleotide sequence is shown in SEQ ID NO. 16 of the sequence listing.

[0023] The method for constructing the above-mentioned glucose dehydrogenase mutant includes the following steps:

[0024] (A1) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues of the glucose dehydrogenase shown in SEQ ID NO.1: E44H (the corresponding amino acid sequence of mutant M1 is shown in SEQ ID NO.2); or

[0025] (A2) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues of the glucose dehydrogenase shown in SEQ ID NO.1: E44H, D176S (corresponding to mutant M2, the amino acid sequence of which is shown in SEQ ID NO.3); or

[0026] (A3) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues of the glucose dehydrogenase shown in SEQ ID NO.1: E44H, D176C (corresponding to mutant M3, the amino acid sequence of which is shown in SEQ ID NO.4); or

[0027] (A4) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues of the glucose dehydrogenase shown in SEQ ID NO.1: E44H, D176E (corresponding to mutant M4, the amino acid sequence of which is shown in SEQ ID NO.5); or

[0028] (A5) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues of the glucose dehydrogenase shown in SEQ ID NO.1: E44H, D176Q (corresponding to mutant M5, the amino acid sequence of which is shown in SEQ ID NO.6); or

[0029] (A6) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues of the glucose dehydrogenase shown in SEQ ID NO.1: E44H, D176S, A253V (corresponding to mutant M6, the amino acid sequence is shown in SEQ ID NO.7); or

[0030] (A7) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues of the glucose dehydrogenase shown in SEQ ID NO.1: E44H, D176S, A253V, K354R (corresponding to mutant M7, the amino acid sequence is shown in SEQ ID NO.8).

[0031] Application of the above glucose dehydrogenase mutants in the synthesis of mannitol and / or gluconic acid.

[0032] Preferably, in the above application, mannitol and / or gluconic acid are synthesized under the combined action of mannitol dehydrogenase, artificial coenzyme NMN and the above glucose dehydrogenase mutant, while the artificial coenzyme NMN(H) is recycled during the synthesis process.

[0033] Preferably, in the above application, gluconic acid is synthesized from glucose as a substrate, while NMNH is regenerated; and mannitol is synthesized from fructose using NMNH as a substrate.

[0034] An artificial coenzyme NMN-oxidoreductase-coenzyme regenerator module is composed of artificial coenzyme NMN, oxidoreductase and the above-mentioned glucose dehydrogenase mutant (as coenzyme regenerator).

[0035] In the application process, an artificial coenzyme NMN-oxidoreductase-coenzyme regenerator module is constructed by adding oxidoreductase and glucose dehydrogenase mutants to artificial coenzyme NMN.

[0036] Preferably, in the above module, the oxidoreductase, as an enzyme utilizing the coenzyme NMNH, is mannitol dehydrogenase or myocardial flavin.

[0037] The modules are: artificial coenzyme NMN-mannitol dehydrogenase-coenzyme regenerating enzyme (the regenerating enzyme is glucose dehydrogenase) module; or artificial coenzyme NMN-myocardial flavonoid-coenzyme regenerating enzyme (the regenerating enzyme is glucose dehydrogenase) module.

[0038] Preferably, in the above module, the glucose dehydrogenase is derived from *Sulfolobus solfataricus*, whose encoding gene is cloned on the pET28a vector.

[0039] Preferably, in the above module, the mannitol dehydrogenase is derived from *Thermotoga maritima*, and its encoding gene is cloned into the pET28a vector.

[0040] Preferably, in the above module, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO.1.

[0041] Preferably, in the above module, the amino acid sequence of the mannitol dehydrogenase is shown in SEQ ID NO.17.

[0042] Preferably, the recipient bacteria used in the above module is Escherichia coli.

[0043] Preferably, in the above module, the myocardial flavonoid is derived from *Geobacillus stearothermophilus*, and its encoding gene is cloned on the pET28a vector.

[0044] Preferably, in the above module, the amino acid sequence of the myocardial flavonoid is shown in SEC ID NO.18.

[0045] The beneficial effects of this invention are:

[0046] The aforementioned glucose dehydrogenase mutant was obtained through saturation mutagenesis, which is a glucose dehydrogenase that responds to the artificial coenzyme NMN. + The mutant with enhanced activity, compared to the wild type, has higher NMN content. +The activity is significantly improved. Mannitol is synthesized using the glucose dehydrogenase mutant described above. The synthesis method involves the catalytic synthesis of mannitol under the combined action of mannitol dehydrogenase, artificial coenzyme, and glucose dehydrogenase mutant, while simultaneously regenerating the artificial coenzyme NMN(H) in the reaction. This method has significant practical value in production.

[0047] This invention provides two artificial coenzyme NMN-oxidoreductase-coenzyme regeneration modules: an artificial coenzyme NMN-mannitol dehydrogenase-coenzyme regeneration enzyme glucose dehydrogenase mutant and an artificial coenzyme NMN-cardiac flavin enzyme-coenzyme regeneration enzyme glucose dehydrogenase mutant. Through a specific combination of the three parts in the module, NMN can be continuously... + Reducing NMNH effectively increases the reaction rate of oxidoreductases and allows coenzymes to be repeatedly recycled, reducing coenzyme consumption and significantly lowering industrial production costs. Attached Figure Description

[0048] Figure 1 The reaction rate of wild-type glucose dehydrogenase at different concentrations;

[0049] Figure 2 A comparison of the specific enzyme activity between glucose dehydrogenase mutant and wild type;

[0050] Figure 3 The reaction rate of mannitol dehydrogenase at different concentrations;

[0051] Figure 4 This is a graph showing the formation process of mannitol and gluconic acid. Detailed Implementation

[0052] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0055] The high-throughput screening principle for glucose dehydrogenase mutants in the following examples is as follows: glucose dehydrogenase catalyzes the synthesis of gluconic acid using glucose as a substrate, while simultaneously generating NMNH; myocardial flavin enzyme uses NMNH to generate a yellow product WST-1 formazan using redox dye WST-1 as a substrate, and potential positive mutants are identified based on the color.

[0056] Example 1

[0057] Cloning and expression of glucose dehydrogenase gene

[0058] A glucose dehydrogenase expression plasmid containing dual promoters T7 and tac was constructed. The constructed prokaryotic expression plasmid was transformed into engineered E. coli bacteria to obtain recombinant strains, and expression of the recombinant strains was induced.

[0059] In this embodiment, glucose dehydrogenase is derived from *Sulfolobus solfataricus*, a sulfur-bearing fungus. The gene is numbered AJ012093.1 on NCBI and can be obtained from the NCBI official website (www.ncbi.nlm.nih.gov / gene / ).

[0060] Construction of pET28a-Ptac-gdh: A new plasmid pET28a-Ptac-gdh was obtained by inserting a tac promoter (5'-ttgacaattaatcatcggctcgtataatgtgt-3') after the T7 promoter of plasmid pET28a-gdh (Xie Leipeng. Construction and optimization of L-lactic acid synthesis pathway using glucose as substrate in vitro multi-enzyme catalysis system [D]. Henan Agricultural University, 2018.). This plasmid contains, in sequence, the T7 promoter, tac promoter, lac operon, RBS, glucose dehydrogenase gene gdh, and pET28a vector backbone. The nucleotide sequence of the wild-type glucose dehydrogenase gene gdh is shown in SEQ ID NO. 9 of the sequence listing, and the amino acid sequence encoding wild-type glucose dehydrogenase GDH is shown in SEQ ID NO. 1 of the sequence listing.

[0061] Ecoli BL21(DE3) carrying the pET28a-Ptac-gdh plasmid was cultured and expressed the recombinant protein GDH. The protein with the His-tag was purified by affinity adsorption, and the activity of the obtained recombinant protein was detected.

[0062] Example 2

[0063] Cloning and expression of myocardial flavin enzyme gene

[0064] The myocardial flavin is derived from the thermophilic bacterium Geobacillus stearothermophilus. The gene is numbered AFD61669.1 on NCBI and can be obtained from the NCBI official website (www.ncbi.nlm.nih.gov / gene / ).

[0065] The pET28a-di plasmid was constructed, which sequentially contains the T7 promoter, lac operon, RBS, myocardial flavin di, and pET28a vector backbone.

[0066] Example 3

[0067] Activity assay of wild-type GDH on NMN+

[0068] GDH and NMN + Preparation for activity assay. The purified enzyme protein was diluted 100,000 times with 50 mM HEPES buffer (pH 7.5) and concentrated using a Millipore 10,000 M CO Amicon centrifuge filter.

[0069] GDH and NMN + The activity assay method. The reaction solution is 100mM HEPES buffer containing 50mM glucose and 20mM NMN. + And 5 mM MgCl2. GDH enzyme activity was measured at 50℃ for 20 min, with an enzyme protein concentration of 0.001-0.005 g / L. GDH can be converted into NMN. + As a coenzyme, GDH catalyzes the production of glucose-1,5-lactone from glucose. Simultaneously, with the increase of NMNH, the generated glucose-1,5-lactone can spontaneously regenerate into gluconic acid. The formation of NMNH was detected using a spectrophotometer, and the catalytic activity of GDH in the production of glucose-1,5-lactone from glucose was calculated. The enzyme activity (U) of GDH is defined as the rate at which GDH catalyzes the production of NMNH from glucose at 50°C per minute. + The amount of enzyme required to generate 1 μmol / L NMNH. Figure 1 The reaction rate of wild-type glucose dehydrogenase at different concentrations is shown. The results indicate that wild-type GDH exhibits the following reaction rates under the above conditions (50℃, 100mM HEPES buffer (pH 7.5), 50mM glucose, 20mM NMN). + With 5 mM MgCl2, the specific enzyme activity was approximately 3.4 U / mg. Figure 1 ).

[0070] Example 4

[0071] High-throughput screening of GDH mutants

[0072] Chemocompetent cells of E. coli T0P10 were prepared using methods described in "Laboratory Guide in Molecular Biology" based on existing technology.

[0073] Recombinant E. coli TOP10 cells carrying the GDH mutant library were cultured on LB solid medium containing 50 μg / mL kanamycin at 37°C for 20 hours to obtain recombinant bacteria. The colonies in the culture dish were heated to 70°C and maintained for 1 hour. Then, 5 mL of a solution containing 0.5% agarose, 50 mM HEPES buffer (pH 7.5), 50 mM glucose, 150 μM WST-1, 3 μg / mL DI, and 2 mM NMN was added. +The chromogenic solution was gently poured onto the heat-treated colonies. After incubation at room temperature for 20 hours, approximately 200 clones were screened on the plates, and GDH-positive mutants were identified by the intensity of the color.

[0074] Example 5

[0075] Enhanced activity of GDH mutant M1

[0076] A GDH mutant library was constructed using site-directed saturation mutagenesis. Using plasmid pET28a-Ptac-gdh as a template, primers were designed (upstream primer: 5′-TGCGGCACCGATCGCNNKATTGTGAACGGCAAA-3′; downstream primer: 5′-TTTGCCGTTCACAATMNNGCGATCGGTGCCGCA-3′), replacing the glutamic acid at position 44 with 19 other amino acids. The mutant product was then transformed into E. coli TOP10 competent cells and plated onto LB agar plates containing 50 μg / mL kanamycin. The cells were incubated overnight at 37°C to obtain recombinant bacteria. Positive mutants were screened using a high-throughput screening method for GDH mutants (as described in *Molecular Biology Laboratory Manual*).

[0077] From the mutants obtained through screening, mutant M1 containing E44H was selected, and its specific enzyme activity was measured. The results showed that the specific enzyme activity of M1 was 5.7 times that of the wild type. Figure 2 ).

[0078] Example 6

[0079] Enhanced activity of GDH mutants M2, M3, M4, and M5

[0080] A GDH mutant library was constructed using site-directed saturation mutagenesis. Using M1 as a template, primers were designed (upstream primer: 5′-GTTTGGACTTGCGATNNKGGCACCCTGAACTGC-3′; downstream primer: 5′-GCAGTTCAGGGTGCCMNNATCGCAAGTCCAAAC-3′), replacing the aspartic acid at position 176 with 19 other amino acids. The mutant product was then transferred into E. coli TOP10 competent cells and plated onto LB agar plates containing 50 μg / mL kanamycin. Recombinant bacteria were obtained by overnight incubation at 37°C. Positive mutants were screened using a high-throughput screening method for GDH mutants (as described in *Laboratory Guide to Molecular Biology*).

[0081] From the mutants obtained through screening, mutant M2 containing D176S, mutant M3 containing D176C, mutant M4 containing D176E, and mutant M5 containing D176Q were selected, and their specific enzyme activities were measured. The results showed that the specific enzyme activity of M2 was 13.6 times that of the wild type; the specific enzyme activity of M3 was 11.1 times that of the wild type; the specific enzyme activity of M4 was 9.4 times that of the wild type; and the specific enzyme activity of M5 was 4.7 times that of the wild type. Figure 2 ).

[0082] Example 7

[0083] Enhanced activity of the GDH mutant M6

[0084] A GDH mutant library was constructed using site-directed saturation mutagenesis. Using M2 as a template, primers were designed (upstream primer: 5′-GATGTGATTATTGATNNKACCGGCGCGGATGTT-3′; downstream primer: 5′-AACATCCGCGCCGGTMNNATCAATAATCACATC-3′), replacing the alanine at position 253 with 19 other amino acids. The mutant product was then transferred into E. coli TOP10 competent cells and plated onto LB agar plates containing 50 μg / mL kanamycin. The cells were incubated overnight at 37°C to obtain recombinant bacteria. Positive mutants were screened using a high-throughput screening method for GDH mutants (as described in *Molecular Biology Laboratory Manual*).

[0085] From the screened mutants, mutant M6 containing A253V was selected, and its specific enzyme activity was measured. The results showed that the specific enzyme activity of M6 was 20 times that of the wild type. Figure 2 ).

[0086] Example 8

[0087] Enhanced activity of GDH mutant M7

[0088] A GDH mutant library was constructed using site-directed saturation mutagenesis. Using M6 as a template, primers were designed (upstream primer: 5′-AAAGTGCTGCGCGAANNKGAACATGGCGAGATC-3′; downstream primer: 5′-GATCTCGCCATGTTCMNNTTCGCGCAGCACTTT-3′), replacing lysine at position 354 with 19 other amino acids. The mutant product was then transferred into E. coli TOP10 competent cells and plated onto LB agar plates containing 50 μg / mL kanamycin. The plates were incubated overnight at 37°C to obtain recombinant bacteria. Positive mutants were screened using a high-throughput screening method for GDH mutants (as described in *Molecular Biology Laboratory Manual*).

[0089] From the mutants obtained through screening, we selected mutant M7 containing K354R and tested the specific enzyme activity of mutant M7. The results showed that the specific enzyme activity of M7 was 20 times that of the wild type. Figure 2 ).

[0090] Gene sequencing was performed on the above mutants, and the specific mutated amino acid sites, as well as the specific amino acid and nucleotide sequences after mutation, are detailed in Table 1.

[0091] Table 1 Mutated amino acid sites

[0092] Mutant name Mutant amino acids amino acid sequence nucleotide sequence M1 E44H SEQ ID NO.2 SEQ ID NO.10 M2 E44H, D176S SEQ ID NO.3 SEQ ID NO.11 M3 E44H, D176C SEQ ID NO.4 SEQ ID NO.12 M4 E44H, D176E SEQ ID NO.5 SEQ ID NO.13 M5 E44H, D176Q SEQ ID NO.6 SEQ ID NO.14 M6 E44H, D176S, A253V SEQ ID NO.7 SEQ ID NO.15 M7 E44H, D176S, A253V, K354R SEQ ID NO.8 SEQ ID NO.16

[0093] Note: The amino acid substitutions in the table are named as follows: original amino acid (wild type), position (i.e., position in SEQ ID NO. 1), substituted amino acid.

[0094] Example 9

[0095] Cloning and expression of mannitol dehydrogenase gene

[0096] Mannitol dehydrogenase is derived from the marine bacterium *Thermotoga maritima*, with the gene number TM0298 on KEGG. The pET28a-mdh plasmid was constructed, which contains the T7 promoter, lac operon, RBS, mannitol dehydrogenase mdh, and pET28a vector backbone.

[0097] Example 10

[0098] Assay of mannitol dehydrogenase (MDH) activity against NMNH

[0099] Preparation for the assay of MDH activity against NMNH. The purified enzyme protein was diluted 100,000 times with 50 mM HEPES buffer (pH 7.5) and concentrated using a Millipore 10,000 MCO Amicon centrifuge filter.

[0100] Method for determining the activity of MDH against NMNH. The reaction solution is 100 mM HEPES buffer (pH 7.5) containing 300 mM fructose, 0.3 mM NMNH, and 5 mM MgCl2. Unless otherwise specified, the MDH enzyme activity assay is performed at 40 °C for 5 min, and the enzyme protein concentration is 0.0025–0.0015 g / L. MDH can catalyze the conversion of fructose to mannitol using NMNH as a coenzyme, accompanied by a decrease in NMNH. The decrease in NMNH is detected using a spectrophotometer, and the catalytic activity of MDH in the conversion of fructose to mannitol is then calculated. The enzyme activity (U) of MDH is defined as the amount of enzyme required for MDH to catalyze the conversion of fructose to 1 μmol / L mannitol per minute at 40 °C. Figure 3 The reaction rate of mannitol dehydrogenase at different concentrations is shown. The results indicate that MDH exhibits a specific enzyme activity of approximately 0.13 U / mg under the above reaction conditions (40℃, 100mM HEPES buffer (pH 7.5), 300mM fructose, 0.3mM NMNH, and 5mM MgCl2).

[0101] Example 11

[0102] GDH and MDH dual-enzyme catalytic reaction

[0103] A 1 mL reaction system contains 500 mM glucose, 500 mM fructose, and 0.1 mM NMN. + The catalytic reaction was carried out at 1 g / L LGDH and 5 g / L LMDH at 40 °C in 200 mM HEPES buffer (pH 7.5) for 48 hours.

[0104] Depending on the retention time, a Sugar-Pak (Waters) RID detector was used to analyze and distinguish gluconic acid, glucose, fructose, or mannitol in the reaction solution. The analytical conditions were: column temperature 80℃, flow rate 0.6 mL / min, mobile phase ultrapure water, and single sample run time 20 min.

[0105] After the reaction was completed, the yield of mannitol was 170 mM and the yield of gluconic acid was 180 mM. Figure 4 ).

[0106] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A glucose dehydrogenase mutant, characterized in that: It is obtained by point mutation of glucose dehydrogenase. The amino acid sequence of glucose dehydrogenase is shown in SEQ ID NO.1 of the sequence listing. The mutation sites are: positions 44, 176, 253 and / or 354 from the N-terminus. Specifically, position 44 from the N-terminus is mutated to E44H, position 176 is mutated to D176S or D176C or D176E or D176Q, position 253 is mutated to A253V, and position 354 is mutated to K354R.

2. The glucose dehydrogenase mutant according to claim 1, characterized in that: include: (I) The nucleic acid molecule encoding the glucose dehydrogenase mutant; (II) An expression cassette, recombinant vector, or recombinant bacteria containing the nucleic acid molecule; The nucleotide sequence of the nucleic acid molecule encoding the glucose dehydrogenase is shown in SEQ ID NO.

9.

3. The glucose dehydrogenase mutant according to claim 1, characterized in that: The mutation site is at position 44 from the N-terminus: E44H, and its amino acid sequence is shown in SEQ ID NO.2 of the sequence listing; more preferably, its nucleotide sequence is shown in SEQ ID NO.10 of the sequence listing; The mutation sites are at positions 44 and 176 from the N-terminus: E44H and D176S, and their amino acid sequences are shown in SEQ ID NO.3 of the sequence listing; more preferably, their nucleotide sequences are shown in SEQ ID NO.11 of the sequence listing. The mutation sites are at positions 44 and 176 from the N-terminus: E44H and D176C, and their amino acid sequences are shown in SEQ ID NO.4 of the sequence listing; more preferably, their nucleotide sequences are shown in SEQ ID NO.12 of the sequence listing. The mutation sites are at positions 44 and 176 from the N-terminus: E44H and D176E, and their amino acid sequences are shown in SEQ ID NO.5 of the sequence listing; more preferably, their nucleotide sequences are shown in SEQ ID NO.13 of the sequence listing. The mutation sites are at positions 44 and 176 from the N-terminus: E44H and D176Q, and their amino acid sequences are shown in SEQ ID NO.6 of the sequence listing; more preferably, their nucleotide sequences are shown in SEQ ID NO.14 of the sequence listing. The mutation sites are at positions 44, 176, and 253 from the N-terminus: E44H, D176S, and A253V, and their amino acid sequences are shown in SEQ ID NO.7 of the sequence listing; more preferably, their nucleotide sequences are shown in SEQ ID NO.15 of the sequence listing. The mutation sites are located at positions 44, 176, 253, and 354 from the N-terminus: E44H, D176S, A253V, and K354R, with amino acid sequences as shown in SEQ ID NO. 8 of the sequence listing; more preferably, their nucleotide sequences are shown in SEQ ID NO. 16 of the sequence listing.

4. The method for constructing the glucose dehydrogenase mutant according to any one of claims 1-3, characterized in that: Includes the following steps: (A1) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues in glucose dehydrogenase: E44H; or (A2) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues in glucose dehydrogenase: E44H, D176S; or (A3) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues in glucose dehydrogenase: E44H, D176C; or (A4) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues in glucose dehydrogenase: E44H, D176E; or (A5) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues in glucose dehydrogenase: E44H, D176Q; or (A6) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues in glucose dehydrogenase: E44H, D176S, A253V; or (A7) The glucose dehydrogenase mutant is a protein obtained by point mutation of the following amino acid residues of glucose dehydrogenase: E44H, D176S, A253V, K354R. The amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO.

1.

5. The use of the glucose dehydrogenase mutant according to any one of claims 1-3 in the synthesis of mannitol and / or gluconic acid.

6. The application according to claim 5, characterized in that: Mannitol and / or gluconic acid are synthesized under the combined action of mannitol dehydrogenase, artificial coenzyme NMN, and glucose dehydrogenase mutant as described in any one of claims 1-3, while the artificial coenzyme NMN(H) is recycled during the synthesis process.

7. The application according to claim 5 or 6, characterized in that: Gluconic acid is synthesized from glucose as a substrate, while NMNH is regenerated; mannitol is synthesized from fructose using NMNH as a substrate.

8. An artificial coenzyme NMN-oxidoreductase-coenzyme regeneration module, characterized in that: It consists of artificial coenzyme NMN, oxidoreductase, and glucose dehydrogenase mutant as described in any one of claims 1-3.

9. The module according to claim 8, characterized in that: The oxidoreductase is mannitol dehydrogenase or myocardial flavin enzyme; preferably, the amino acid sequence of the mannitol dehydrogenase is shown in SEQ ID NO.17; and the amino acid sequence of the myocardial flavin enzyme is shown in SEQ ID NO.

18.

10. The module according to claim 8 or 9, characterized in that: The gene encoding glucose dehydrogenase is cloned into the pET28a vector, the gene encoding mannitol dehydrogenase is cloned into the pET28a vector, and the gene encoding myocardial flavonoids is cloned into the pET28a vector.

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