Glucose dehydrogenase mutant and its application

Abstract

The present invention relates to the field of enzyme engineering technology, and in particular to glucose dehydrogenase mutants and applications thereof. The present invention discloses a glucose dehydrogenase mutant, wherein the glucose dehydrogenase mutant is selected from mutations at at least one of the amino acids at positions 20, 132, 135, 176 and 182 of the amino acid sequence shown in SEQ ID NO.1. The glucose dehydrogenase mutant provided by the present invention has a higher reductase activity for NMN than the conventional wild-type glucose dehydrogenase mutant, and its enzyme activity can reach 1220 times that of the wild type, and can efficiently reduce NMN to NMNH; the glucose dehydrogenase mutant can be applied more than 20 times after being immobilized, and 800mM NMN can be completely converted within 1 hour, and 1.2M NMN can be completely converted within 1.5 hours.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, in particular to a glucose dehydrogenase mutant and application thereof. Background Art

[0002] NMNH, full name reduced β-nicotinamide mononucleotide, is the direct precursor of NADH (reduced nicotinamide adenine dinucleotide) and is a derivative of vitamin B3 (nicotinic acid or niacin). As a direct precursor of NADH, NMNH participates in multiple metabolic pathways in cells, including glycolysis, tricarboxylic acid cycle (TCA cycle), fatty acid oxidation, etc.

[0003] In cells, NMNH can be synthesized through a variety of pathways. One of the main pathways is through the reduction reaction of NMN (β-nicotinamide mononucleotide). The synthesis of NMNH is strictly regulated by the redox state in cells. NMN reductase (NMNR) is the key enzyme that catalyzes the conversion of NMN to NMNH. Changes in the expression of NMN reductase in cells will affect the ratio of NADH / NAD+, thereby affecting the metabolic state and energy balance of cells.

[0004] In the prior art, NMNH can be prepared by, for example, the following chemical synthesis method or enzymatic synthesis method.

[0005] 1. Chemical synthesis of NMNH

[0006] 10% ammonia water (v / v) and 1.2 equivalents of dithiourea were added to the NMN solution, and the reaction was stirred at 40°C and pH 9.0 for 1 hour. The NMN conversion rate was greater than or equal to 90%.

[0007] 2. Enzymatic synthesis of NMNH

[0008] (1) Using 5 mM reduced nicotinamide adenine dinucleotide (NADH) as the raw material, MnCl2 and NADH pyrophosphatase were added, and the reaction was stirred at pH 8.0 and 37°C for 45 min. The conversion rate of NADH was about 70%;

[0009] (2) Using 100 mM NMN and 200 mM formic acid as raw materials, adding formate dehydrogenase, and reacting at pH 7.0-8.0 and 30°C for 4 h, the NMN conversion rate can reach up to 69.8%.

[0010] The cost of NMNH synthesized by the above two methods is relatively high, which limits its market prospects. Summary of the invention

[0011] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a glucose dehydrogenase mutant having high reductase activity for NMN and its application.

[0012] In a first aspect of the present application, a glucose dehydrogenase mutant is provided, wherein the glucose dehydrogenase mutant is selected from mutations at at least one of the amino acids at positions 20, 132, 135, 176 and 182 of the amino acid sequence shown in SEQ ID NO.1.

[0013] In some embodiments of the present invention, the amino acid sequence as shown in SEQ ID NO.1 is mutated at position 20 to at least one of lysine, arginine, leucine, and alanine;

[0014] The amino acid sequence as shown in SEQ ID NO.1 is mutated at position 132 to at least one of threonine, tyrosine and alanine;

[0015] The amino acid sequence as shown in SEQ ID NO.1 is mutated at position 135 to at least one of asparagine, leucine, glutamic acid and alanine;

[0016] The amino acid sequence as shown in SEQ ID NO.1 is mutated at position 176 to at least one of valine, aspartic acid, isoleucine, asparagine and serine;

[0017] The amino acid sequence shown in SEQ ID NO.1 is mutated at position 182 to at least one of arginine, isoleucine, threonine, glutamic acid and alanine.

[0018] In some embodiments of the present invention, the amino acid sequence of the glucose dehydrogenase mutant is shown in any one of SEQ ID NO.2 to SEQ ID NO.11;

[0019] The second aspect of the present application provides a nucleic acid molecule, the nucleotide sequence of the nucleic acid molecule is (A1) or (A2):

[0020] (A1) a nucleotide sequence encoding the above-mentioned glucose dehydrogenase mutant;

[0021] (A2) A nucleotide sequence complementary to the nucleotide sequence described in (A1).

[0022] The third aspect of the present application provides a vector, which can express the glucose dehydrogenase mutant described in the first aspect of the present invention or contains the nucleic acid molecule described in the second aspect of the present invention.

[0023] In some embodiments of the present invention, the vector is at least one of pET, pCW, pUC or pPIC9k.

[0024] The fourth aspect of the present application provides a recombinant cell comprising the vector described in the third aspect of the present application, wherein the recombinant cell is not a new variety of animal or plant.

[0025] In some embodiments of the present invention, the recombinant cell is selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Streptomyces, Bacillus subtilis and other eukaryotic or prokaryotic cells well known in the art that can be used to express the target protein.

[0026] The fifth aspect of the present invention provides the use of the glucose dehydrogenase mutant described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the recombinant cell described in the fourth aspect of the present invention in (B1) or (B2):

[0027] (B1) Catalytic synthesis of NMNH;

[0028] (B2) Preparation of products of catalytic synthesis of NMNH.

[0029] The sixth aspect of the present invention provides a product comprising the glucose dehydrogenase mutant described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the recombinant cell described in the fourth aspect of the present invention.

[0030] The seventh aspect of the present invention provides a method for preparing a glucose dehydrogenase mutant: the recombinant cell described in the fourth aspect of the present invention is cultured to obtain a culture, and the glucose dehydrogenase mutant is isolated from the culture.

[0031] In some embodiments of the present invention, after the crude enzyme solution of the glucose dehydrogenase mutant is separated, it can be further purified to obtain the glucose dehydrogenase mutant with higher purity.

[0032] An eighth aspect of the present invention provides a method for preparing NMNH, comprising the following steps:

[0033] Provide glucose and NMN as substrates;

[0034] The substrate is contacted with the above-mentioned glucose dehydrogenase mutant to catalyze the synthesis of NMNH.

[0035] Among them, providing glucose and NMN as substrates means directly providing glucose and NMN, and under the catalysis of the glucose dehydrogenase mutant, glucose and NMN undergo a redox reaction to generate gluconolactone and NMNH. In addition, other reaction aids or reaction conditions required in the catalytic synthesis process can be adjusted accordingly according to the optimal reaction temperature and other conditions of the enzyme used in the corresponding synthesis step of the reaction process.

[0036] In some embodiments of the present invention, the method for preparing NMNH comprises the following steps:

[0037] (a) adding a solution containing a substrate into a reactor, and adjusting the pH of the solution to 7.0 to 10.0;

[0038] (b) adding glucose dehydrogenase to the solution to obtain a reaction system;

[0039] (c) controlling the system temperature at 30 to 40° C. and maintaining the pH of the reaction system between 7.0 and 10.0 to carry out the catalytic reaction;

[0040] (d) obtaining a crude solution containing NMNH;

[0041] (e) The crude solution is filtered, purified, and dried to obtain NMNH.

[0042] Among them, the glucose dehydrogenase mutant and some other enzymes required in the reaction process participate in the catalytic reaction in at least one form of enzyme solution, enzyme lyophilized powder, enzyme-containing cells, immobilized enzyme or immobilized enzyme cells.

[0043] The beneficial effects of the present invention are:

[0044] The glucose dehydrogenase mutant provided by the present invention has a higher reductase activity for NMN than the conventional wild-type glucose dehydrogenase mutant, and its enzyme activity can reach 1220 times that of the wild type, and can efficiently reduce NMN to NMNH; the glucose dehydrogenase mutant can be applied more than 20 times after immobilization, and can completely convert 800mM NMN within 1 hour, and 1.2M NMN can be completely converted within 1.5 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0046] Figure 1 HPLC chart of the product NMNH when the immobilized enzyme reaction is terminated in Example 6;

[0047] Figure 2 This is the HPLC chart of the substrate NMN at the start of the immobilized enzyme reaction in Example 6;

[0048] Figure 3 HPLC charts of NMNH reference substance (data 1) and product NMNH (data 2);

[0049] Wherein: the NMNH reference substance is the standard reference sample of NMNH; the product NMNH is the product NMNH prepared in Example 6. DETAILED DESCRIPTION

[0050] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments, and the contents mentioned in the implementation modes are not intended to limit the present invention.

[0051] As used herein, "and / or" includes the term of any and all combinations of one or more associated listed items. The terms used herein are only used to describe specific embodiments and are not intended to limit the present invention. As used herein, the singular forms "a", "an", "an" and "the" are also intended to include the plural forms, unless the context clearly indicates otherwise. It is further understood that "including", when used in this specification, specifies the stated features, integers, steps, operations, elements and / or compositions, but does not exclude the existence or addition of one or more other features, integers, steps, operations, elements, compositions and / or combinations thereof.

[0052] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It is further understood that terms, such as defined in commonly used dictionaries, are interpreted in accordance with their meanings in the context of the relevant art and are not idealized or overly formal meanings unless explicitly defined as such herein.

[0053] The exemplary invention described herein may appropriately lack any one or more element limitations that are not specifically disclosed herein. Therefore, the terms "comprise", "include", "contain", etc. should be understood broadly and non-restrictively. In addition, the terminology used herein is used as a description, not a limitation, and it is unintentional to use these terminology expressions that do not include any equivalent characteristics, but only describe some of their characteristics, but various modifications are possible within the scope of the present invention according to the rights. Therefore, although the present invention has been specifically disclosed by preferred embodiments and optional features, the modifications disclosed herein to embody the changes of the present invention may be recorded by those skilled in the art, and such modifications and changes will be considered to be within the scope of the present invention.

[0054] Example 1

[0055] 1. Gene acquisition

[0056] 1. Search the amino acid sequence of enzyme number EC 1.1.1.118 (NAD converted to NADH) from the NCBI database, remove the sequence with EC 1.1.1.47 enzyme function, and finally find the amino acid sequence numbered 2DTX from Thermoplasma acidophilum through the PDB database;

[0057] The amino acid sequence is shown in SEQ ID NO.1:

[0058] MGFSDLRDKVVIVTGASMGIGRAIAERFVDEGSKVIDLSIHDPGEAKYDHIECDVTNPDQVKASIDHIFKEYGSISVLVNNAGIESYGKIESMSMGEWRIIDVNLFGYYYASKFAIPYMIRSRDPSIV NISSVQASIITKNASAYVTSKHAVIGLTKSIALDYAPLLRCNAVCPATIDTPLVRKAAELEVGSDPMRIEKKISEWGHEHPMQRIGKPQEVASAVAFLASREASFITGTCLYVDGGLSIRAPISTP (SEQ ID NO.1).

[0059] 2. Codon optimization was performed, and the optimized nucleotide sequence is shown in SEQ ID NO.12:

[0060] (SEQ ID NO.12);

[0061] The vector was transformed into E. coli BL21(DE3) strain by digestion with NdeI and EcoRI and ligated to pET-22b (+). After correct sequencing, the TaGDH-pET22b strain was obtained.

[0062] 3. Through reaction solution 1 (10mM NAD+20mM glucose) and reaction solution 2 (10mM NADP+20mM glucose), it was determined that the biological enzyme only has the function of catalyzing NAD to produce NADH.

[0063] Example 2

[0064] 2. Selection of mutation sites

[0065] 1. The three-dimensional structures of the substrate NMN, product NMNH and amino acid sequence were molecularly docked by AUTODOCK. Based on the preliminary results, the active pockets of the amino acid sequence were determined. After screening, the first round of mutation sites were determined to be I20A, S132A, Q135A, A176S, and T177A;

[0066] 2. The first round of mutagenesis primers are as follows:

[0067] I20A-F: 5'-GTGCCAGCATGGGTGCGGGTCGCGCAATTG-3';

[0068] I20A-R: 5'- CAATTGCGCGACCCGCACCCATGCTGGCAC-3';

[0069] S132A -F: 5'-GTATTGTTAATATTGCCAGTGTGCAGGCC -3';

[0070] S132A -R: 5'- GGCCTGCACACTGGCAATATTAACAATAC -3';

[0071] Q135A -F: 5'- TATTAGCAGTGTGGCGGCCAGTATTATTAC -3';

[0072] Q135A -R: 5'- GTAATAATACTGGCCGCCACACTGCTAATA -3';

[0073] A176S -F: 5'- GCAGTTTGTCCGTCAACCATTGATACACC -3';

[0074] A176S -R: 5'- GGTGTATCAATGGGTTGACGGACAAACTGC -3';

[0075] T177A -F: 5'-GTTTGTCCGGCAGCCATTGATACACCTCTG -3';

[0076] T177A -R: 5'-CAGAGGTGTATCAATGGCTGCCGGACAAAC -3';

[0077] 3. Mutation process

[0078] 1) Use KOD OneTM PCR Master Mix from Toyobo for whole plasmid site-directed mutagenesis PCR;

[0079] 2) PCR system

[0080]

[0081] 3) PCR procedure

[0082]

[0083] 4) DpnI digestion

[0084] After the PCR reaction, 1 µl of DpnI (Full Gold DMT Enzyme (DpnI)) was directly added to the PCR reaction system, mixed and incubated at 37°C for 15 min, and heated at 80°C for 20 min for inactivation;

[0085] 5) Conversion

[0086] After Dpnl digestion, it was directly transformed into DH5a: 5µl of the mutant product after Dpnl digestion was added to 100µl of DH5a chemically competent cells, mixed and placed on ice for 30 minutes; heat shock at 42°C for 90 seconds, then placed on ice for 2 minutes, added LB liquid culture medium, and cultured at 37°C with shaking for 1 hour; finally, LB solid plates with ampicillin resistance were used for coating and cultured in a 37°C incubator overnight.

[0087] 6) Select single clones and sequence them;

[0088] Five clones were selected for each mutation site, inoculated into liquid culture medium containing ampicillin and cultured for 8 h. After that, the plasmids were extracted and sequenced for verification.

[0089] 4. The verified correct plasmid was transformed into E. coli BL21 (DE3) strain, and the obtained TaGDH single mutant strains were named TaGDH-I20A, TaGDH-S132A, TaGDH-Q135A, TaGDH-A176S, and TaGDH-T177A.

[0090] Example 3

[0091] 3. Enzyme activity determination

[0092] 1. Obtaining enzyme solution

[0093] 1) Seed liquid activation: Inoculate 1% of the wild-type and mutant E. coli BL21 (DE3) strain glycerol culture into LB liquid culture medium containing 100 mg / L ampicillin and culture overnight (16 h) in a shaking incubator at 37°C.

[0094] 2) Shake flask culture: add 1% of the seed activation solution cultured overnight to the culture medium containing the corresponding antibiotics, and culture it in a 37℃ constant temperature rotary shaker for about 3 hours until the OD600 is 0.6-0.8. Add IPTG inducer to a final concentration of 1mM. Place it in a 25℃ shaker at 200rpm and continue to culture for 16 hours.

[0095] 3) Collection of bacterial sludge and preparation of enzyme solution: Collect bacterial sludge by centrifugation at 4℃ 10000rpm for 10min, add 100mM PBS (pH7.6) solution at the ratio of bacterial sludge weight: PBS solution = 1g:4mL for resuspending, and after ultrasonic disruption, centrifuge at 4℃ 10000rpm for 10min, collect the supernatant as the enzyme solution.

[0096] 2. Enzyme activity determination method

[0097] The total protein content of the enzyme solution was determined according to the Coomassie Brilliant Blue G250 method, and the enzyme solution was diluted to a protein concentration of 15 mg / ml;

[0098] Take 200µl of the diluted enzyme solution and add it to 800ul of reaction solution (100mM NMN+100mM glucose, pH 8.0±0.5), place it in a 30℃ water bath oscillator at 100rpm for 10min, take a sample (dilute with 0.1M sodium bicarbonate solution, filter through 0.22µm and then perform liquid HPLC to detect the sample)

[0099] 3. Enzyme activity results

[0100] The amount of enzyme required to convert 1 μmol NMN to NMNH in 1 min is defined as 1 U. The activity of each enzyme is calculated and the results are as follows:

[0101]

[0102] Example 4

[0103] 4. Second round of mutation

[0104] 1. Introduce new mutations and perform saturation mutation:

[0105] I20-F: 5'-GTGCCAGCATGGGTNNNKCGCGCAATTG-3';

[0106] I20-R: 5'-CAATTGCGCGACCMNNCATGCTGGCAC-3';

[0107] S132-F:

[0108] 5'- CCGAGTATTGTTAATATTNNKAGTGTGCAGGCCAG -3';

[0109] S132-R:

[0110] 5'-CTGGCCTGCACACTMNNAATATTAACAATACTCGG -3';

[0111] Q135-F: 5'- TATTAGCAGTGTGNNKGCCAGTATTATTAC -3';

[0112] Q135-R: 5'- GTAATAATACTGGCMNNCACACTGCTAATA -3';

[0113] A176-F: 5'-GCAGTTTTGTCCGNNKACCATTGATACACC -3';

[0114] A176-R: 5'- GGTGTATCAATGGTMNNCGGACAAACTGC -3';

[0115] L182-F: 5'- CCATTGATACACCTNNKGTGCGTAAAGCAG -3';

[0116] L182-R: 5'-CTGCTTTACGCACMNNAGGTGTATCAATGG -3';

[0117] 2. Enzyme activity results

[0118] 1) Enzyme activity of saturated mutation (only mutation sites with enzyme activity are recorded)

[0119]

[0120] Example 5

[0121] 5. Multi-site mutation

[0122] 1. Based on the results of the first and second rounds of mutation, multi-site mutation was performed, and the primers were as follows:

[0123] S132Y-F:

[0124] 5'- CCGAGTATTGTTAATATTTACAGTTGTGCAGGCCAG -3';

[0125] S132Y-R:

[0126] 5'-CTGGCCTGCACACTGTAAATATTAACAATACTCGG -3';

[0127] Q135E-F: 5'- TATTAGCAGTGTGGAGGCCAGTATTATTACC -3';

[0128] Q135E-R: 5'- GGTAATAATACTGGCCTCCACACTGCTAATA -3';

[0129] L182R-F: 5'-CATTGATACACCTCCGGTGCGTAAAGCAGC-3';

[0130] L182R-R: 5'-GCTGCTTTACGCACCGGAGGTGTATCAATG -3';

[0131] 2. Obtained mutant strains and enzyme activity results:

[0132]

[0133] SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11 are detailed in the sequence listing.

[0134] Example 6

[0135] 6. Immobilization of biological enzymes

[0136] 1. The enzyme solution of the mutant TaGDH-I20K / A176S / S132Y / Q135E / L182R was affinity adsorbed by Xi'an Lanxiao's NiSeplife FF (IDA) chromatography medium, and then covalently cross-linked using Xi'an Lanxiao's SEPLITE® LX-1000EP resin. The protein concentration was tested and the protein loading of the immobilized enzyme was confirmed to be 37.52 mg / g.

[0137] 2. Prepare 2 10 ml reaction solutions with a final concentration of 100 mM NMN + 120 mM glucose, adjust the pH to 8.0, add enzyme solution with a final protein concentration of 2 g / L and immobilized enzyme, respectively, and place in a 30°C water bath oscillator at 100 rpm for reaction; experimental results (expressed as the conversion rate of substrate NMN):

[0138]

[0139] Example 7

[0140] 7. Optimization of reaction conditions

[0141] 1. Reaction pH gradient experiment

[0142] 1) Prepare 5 50 ml reaction solutions with a final concentration of 100 mM NMN and 120 mM glucose, adjust the pH to 8.0, 8.5, 9.0, 9.5, and 10.0, respectively, take samples, add 1 g of immobilized enzyme (final concentration 0.75 g / L) to each, place in a 30°C water bath oscillator at 100 rpm for reaction, and the reaction results (expressed as the conversion rate of substrate NMN) are:

[0143]

[0144] 2) Based on the experimental results of the above experiment 1), pH 9.5 was selected as the pH value of the reaction, and a repetitive experiment was performed to monitor the conversion rate of the substrate NMN over the reaction time:

[0145]

[0146] 2. Reaction temperature gradient experiment

[0147] 1) Prepare 5 50 ml reaction solutions with a final concentration of 100 mM NMN and 120 mM glucose, adjust the pH to 9.5, take samples, add 1 g of immobilized enzyme (final concentration 0.75 g / L) to each, and place in a water bath oscillator at 20, 25, 30, 35, and 40 ° C at 100 rpm for reaction. The reaction results (expressed as the conversion rate of substrate NMN) are:

[0148]

[0149] 2) When the reaction temperature is greater than 30°C, the conversion rate of the substrate NMN is similar. Considering that the degradation rate of the substrate NMN under alkaline conditions will increase with increasing temperature, 30°C is selected as the optimal reaction temperature;

[0150] 3. NMN concentration gradient

[0151] 1) Prepare 7 50 ml reaction solutions with NMN concentrations of 100, 200, 400, 600, 800, 1000, and 1200 mM, respectively. The glucose concentration is 1.2 equivalents of the NMN concentration. Adjust the pH to 9.5±0.2 and take samples. Add 1 g of immobilized enzyme (final concentration 0.75 g / L) to each solution. Place the solution in a 30°C water bath oscillator at 100 rpm for the reaction. The reaction results (expressed as the conversion rate of the substrate NMN) are as follows:

[0152]

[0153] 2) Based on the reaction time and reaction results, considering that the reaction time was controlled within 60 min, 800 mM was selected as the NMN concentration for subsequent experiments;

[0154] 4. Glucose addition amount

[0155] 1) Prepare 300 ml of reaction solution, NMN concentration 800 mM, adjust pH to 9.5 ± 0.2, and take samples; take 50 ml of reaction solution, add glucose with molar concentration of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 equivalents, add 2 g of immobilized enzyme (final concentration 1.5 g / L), place in a 30°C water bath oscillator at 100 rpm for reaction, the reaction results (expressed as the conversion rate of substrate NMN):

[0156]

[0157] 2) Based on the experimental results and cost considerations, the amount of glucose added was selected to be 1.2 equivalents, that is, 960 mM glucose was added to the 800 mM NMN reaction solution;

[0158] 5. Addition amount of immobilized enzyme

[0159] 1) Prepare 5 50 ml reaction solutions with a final concentration of 800 mM NMN and 960 mM glucose, adjust the pH to 9.5 ± 0.2, take samples, add 1.0, 1.5, 2.0, 2.5, 3.0 g of immobilized enzyme (final protein concentrations of 0.75, 1.125, 1.5, 1.875, 2.25 g / L, respectively), place in a 30°C water bath oscillator at 100 rpm for reaction, the reaction results (expressed as the conversion rate of substrate NMN):

[0160]

[0161] 2) To ensure that the reaction is complete within 1 hour, 2.0 g of immobilized enzyme was added to 50 ml of reaction solution (final concentration 1.5 g / L) as the feeding standard for the immobilized enzyme.

[0162] Example 8

[0163] 8. Immobilized enzyme application experiment

[0164] 1. Prepare 500 ml of reaction solution with a final concentration of 800 mM NMN and 960 mM glucose, adjust the pH to 9.5 ± 0.2, take a sample and divide it into 20 portions, each 25 ml. Perform 20 replicate experiments with 1.0 g of immobilized enzyme (final concentration 1.5 g / L).

[0165] 2. Experimental results (reaction for 1 hour): The results of the 1st to 17th application experiments all showed that the conversion rate of substrate NMN was 100%; after the 18th application, the enzyme activity decreased, and the conversion rate of substrate NMN was 99.92%; after the 19th application, the conversion rate of substrate NMN was 99.94%; after the 20th application, the conversion rate of substrate NMN was 99.90%.

[0166] Example 9

[0167] 9. Hundred-gram Experiment

[0168] 1. Prepare 3 500ml reaction solutions with a final concentration of 800mM NMN and 960mM glucose, adjust the pH to 9.5±0.2, add 20g of immobilized enzyme (final protein concentration 1.5g / L) to each, place in a 30℃ water bath and stir with a mechanical stirrer at 100rpm for 1 hour. Experimental results:

[0169]

[0170] 2. Hundred-gram-scale experiments confirmed that the immobilized enzyme still had the characteristic of completely converting the substrate NMN within 1 hour in the scale-up experiment.

[0171] The raw materials or reagents used in the examples and comparative examples of the present invention are all purchased from mainstream manufacturers in the market. Those without indicating the manufacturer or concentration are all analytically pure raw materials or reagents that can be routinely obtained. As long as they can play the expected role, there is no special limitation.

[0172] The instruments and equipment used in this embodiment are all purchased from major manufacturers in the market, and there is no particular limitation as long as they can play the expected role. In this embodiment, if no specific technology or conditions are specified, the technology or conditions described in the literature in this field or the product instructions are used. The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention can also be implemented in other ways. Any obvious replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A glucose dehydrogenase mutant, characterized in that: The glucose dehydrogenase mutant is selected from the amino acid sequence shown in SEQ ID NO.1, wherein the amino acid position 176 is mutated from alanine to serine.

2. A glucose dehydrogenase mutant, characterized in that: The amino acid sequence of the glucose dehydrogenase mutant is shown in any one of SEQ ID NO.2 to SEQ ID NO.

11.

3. A nucleic acid molecule, characterized in that The nucleotide sequence of the nucleic acid molecule is (A1): (A1) A nucleotide sequence encoding the glucose dehydrogenase mutant according to any one of claims 1 to 2.

4. A carrier, characterized in that The vector can express the glucose dehydrogenase mutant according to any one of claims 1 to 2 or contain the nucleic acid molecule according to claim 3.

5. A recombinant cell, characterized in that The recombinant cell comprises the vector of claim 4.

6. Use of the glucose dehydrogenase mutant according to any one of claims 1 to 2, the nucleic acid molecule according to claim 3, the vector according to claim 4, or the recombinant cell according to claim 5 in (B1) or (B2): (B1) catalyzes the synthesis of reduced β-nicotinamide mononucleotide; (B2) preparing a product for catalytic synthesis of reduced β-nicotinamide mononucleotide.

7. A product having reductase activity on β-nicotinamide mononucleotide, characterized in that: Comprising the glucose dehydrogenase mutant according to any one of claims 1 to 2, or the nucleic acid molecule according to claim 3, or the vector according to claim 4, or the recombinant cell according to claim 5.

8. A method for preparing a glucose dehydrogenase mutant, characterized in that: The glucose dehydrogenase mutant is obtained by culturing the recombinant cell according to claim 5.

9. A method for preparing reduced β-nicotinamide mononucleotide, characterized in that: The following steps are involved: β-nicotinamide mononucleotide and glucose were provided as substrates; The substrate is contacted with the glucose dehydrogenase mutant according to any one of claims 1 to 2 to catalyze the synthesis of the reduced β-nicotinamide mononucleotide.

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