Glucose 6-phosphate dehydrogenase mutant with NMN < + > preference and preparation method thereof

Abstract

The invention discloses a glucose 6-phosphate dehydrogenase mutant with NMN < + > preference and a preparation method of the glucose 6-phosphate dehydrogenase mutant, and belongs to the field of gene engineering and enzyme engineering. According to the present invention, the existing TmG6PDH-R7 mutant is adopted as the starting protein, the error-prone PCR technology is adopted to carry out random mutation, the random mutant library is constructed, the plate positive clone high-throughput screening method is adopted to obtain the clone with the most obvious color development, the recombinant bacteria are cultured, and the purification is performed to obtain the mutant; through three rounds of screening, the glucose 6-phosphate dehydrogenase mutant with enhanced NMN < + > preference as shown in the amino acid sequence of SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.10 is finally obtained, the specific enzyme activity of TmG6PDH with NMN < + > as a coenzyme is remarkably improved, and compared with a currently known R7 mutant with the highest specific enzyme activity, the specific enzyme activity of the mutant with the amino acid sequence as shown in SEQ ID NO.10 is improved by more than 5 times at 37 DEG C, and the specific enzyme activity of the mutant with the amino acid sequence as shown in SEQ ID NO.8 or SEQ ID NO.10 is remarkably improved. The method has a good application prospect in industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and enzyme engineering, and relates to a method for + A preferential glucose 6-phosphate dehydrogenase mutant, specifically one that can synthesize NMN as a biomimetic coenzyme + A glucose 6-phosphate dehydrogenase mutant used as a cofactor to carry out redox reaction and a preparation method thereof. Background Art

[0002] NAD(P) + Oxidoreductases, as natural coenzymes, are widely used in biocatalytic reactions that can replace traditional organic synthesis. However, the high cost and instability of natural coenzymes limit the large-scale application of these biocatalytic reactions. Natural coenzyme NAD(P) + It consists of two parts: the nicotinamide region and the phosphorylated or non-phosphorylated adenosine dinucleotide. The former is responsible for electron transfer, while the latter is responsible for recognition and positioning in the enzyme active center. Biomimetic cofactors are mainly based on nicotinamide as the parent nucleus and are formed by replacing the adenosine dinucleotide in the natural coenzyme with some relatively stable structures such as sugar, phenyl or benzyl. Reported biomimetic cofactors include NFCD + (nicotinamide flucytosinedinucleotide), NR +

[0003] (nicotinamide mononucleoside), NMN + (nicotinamide mononucleotide) and BNA + (1-benzyl nicotinamide), etc. Using highly stable, low-cost biomimetic coenzymes to replace unstable and expensive natural coenzymes is an effective way to reduce the cost of large-scale biocatalytic applications. However, the catalytic ability of redox enzymes to utilize biomimetic coenzymes and the regeneration of reduced biomimetic coenzymes remain pressing challenges.

[0004] In the in vitro multi-enzyme molecular machine catalyzing the production of hydrogen and bioelectricity from monosaccharides and polysaccharides, coenzymes are the key to mediating the transfer of electrons and protons to generate products. +Preference transformation has important application value.

[0005] Recent studies have significantly improved the ability of these enzymes to utilize the biomimetic coenzyme NMN through multiple rounds of directed evolution. + The ability to perform redox reactions. Among them, the glucose 6-phosphate dehydrogenase mutant (TmG6PDH-R7) is based on NMN + The highest specific enzyme activity was 2.8 U mg -1 (60℃), which is the highest level reported so far, but still much lower than that based on NADP + The specific enzyme activity of wild-type glucose 6-phosphate dehydrogenase (14 U mg -1 ). Therefore, in the + In the industrial production of hydrogen and bioelectricity using an in vitro multi-enzyme system, further NMN characterization of glucose 6-phosphate dehydrogenase is urgently needed. + Preferential modification to achieve complete replacement of natural coenzyme NADPH with bionic coenzyme NMN. Summary of the Invention

[0006] The purpose of the present invention is to provide a biomimetic coenzyme NMN + Preferential glucose 6-phosphate dehydrogenase mutants and preparation methods thereof, by conducting extensive mutation screening based on existing mutants, ultimately obtained mutants with significantly improved enzyme activity.

[0007] The present invention is achieved through the following technical solutions:

[0008] Using TmG6PDH-R7, whose amino acid sequence is shown in SEQ ID NO. 4, as the starting protein, which had undergone thermostability and primary directed evolution, the gene was randomly mutated using error-prone PCR technology, and positive clones were screened using a high-throughput plate-based positive clone screening method.

[0009] The NMN + The preferred glucose 6-phosphate dehydrogenase mutants are as follows:

[0010] (a) First round of transformation: Using TmG6PDH-R7 (SEQ ID NO. 4) as the starting protein, random mutations were performed by error-prone PCR to construct a library of glucose 6-phosphate dehydrogenase mutants. A high-throughput screening method for plate-positive clones was used to obtain NMN encoding proteins. + A nucleic acid encoding a polypeptide of a glucose 6-phosphate dehydrogenase mutant with enhanced preference, wherein the protein sequence encoded by the nucleic acid is shown in SEQ ID NO. 6 (R8);

[0011] (b) Second round of transformation: Using the optimal mutant R8 from the first round as the starting protein, random mutations were performed using error-prone PCR to construct a library of glucose 6-phosphate dehydrogenase mutants. A high-throughput screening method for plate-positive clones was used to obtain NMN-encoding proteins. + A nucleic acid encoding a polypeptide of a glucose 6-phosphate dehydrogenase mutant with enhanced preference, wherein the protein sequence encoded by the nucleic acid is shown in SEQ ID NO. 8 (R9);

[0012] (c) The third round of transformation: Using the optimal mutant R9 from the second round as the starting protein, random mutations were performed using error-prone PCR technology to construct a library of glucose 6-phosphate dehydrogenase mutants. A high-throughput screening method for plate-positive clones was used to obtain the NMN encoding + A nucleic acid encoding a polypeptide of a glucose 6-phosphate dehydrogenase mutant with enhanced preference, wherein the protein sequence encoded by the nucleic acid is shown in SEQ ID NO. 10 (R10);

[0013] In summary, after three rounds of transformation, we obtained NMN + A preferred glucose 6-phosphate dehydrogenase mutant, the amino acid sequence of which is shown in SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.10.

[0014] The present invention also provides a method for encoding the NMN + The gene of the preferential glucose 6-phosphate dehydrogenase mutant has a nucleotide sequence as shown in SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.9.

[0015] The present invention also provides a vector or a cell carrying the gene.

[0016] Except for the above mutants R8, R9, and R10, other mutants with NMN + The preferred glucose 6-phosphate dehydrogenase mutant has an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 6, SEQ ID NO. 8, and SEQ ID NO. 10, and in particular at least 90% homologous.

[0017] The present invention also provides a method having NMN + The method for preparing a preferential glucose 6-phosphate dehydrogenase mutant comprises the following steps:

[0018] Using a vector containing the gene encoding the TmG6PDH mutant (pET28a-Ptac-tmg6pdh-R7 in the present embodiment) as a template, random mutagenesis was performed using error-prone PCR to construct a library of glucose 6-phosphate dehydrogenase mutants. A high-throughput screening method for plate-positive cloning was used to obtain one or more clones with the most obvious color development. Plasmids were then extracted and sequenced for mutation analysis. The vector carrying the mutant gene was introduced into Escherichia coli BL21(DE3), and the recombinant bacteria were cultured. Glucose 6-phosphate dehydrogenase mutants were obtained by heat treatment or nickel column purification.

[0019] In some embodiments, the gene encoding the TmG6PDH mutant is a sequence as shown in SEQ ID NO.3, or a sequence as shown in SEQ ID NO.11 is added to the 5' end of the sequence as shown in SEQ ID NO.3.

[0020] The nucleotide sequence encoding wild-type glucose 6-phosphate dehydrogenase is shown in SEQ ID NO. 1, which is derived from Thermotoga maritima and synthesized from the sequence with UniProt accession number Q9X0N9 after codon optimization in Escherichia coli. The reference is Li, Qiangzi, et al. "Stoichiometric Regeneration of Biomimetic Nicotinamide Coenzyme Powered by Biomass Sugars via In VitroSynthetic Enzymatic Biosystems." ChemSusChem 18.4(2025):

[0021] e202401263.

[0022] The beneficial effects of the present invention are as follows:

[0023] After three rounds of random mutant library construction and high-throughput screening, 3 NMNs were obtained. + TmG6PDH mutants with enhanced preference all achieved NMN + The enzyme activity of TmG6PDH as a coenzyme was significantly improved. In particular, compared with the R7 mutant with the highest known enzyme activity, the R10 mutant screened out had an enzyme activity at 37 degrees that was increased by more than 5 times. + Preferential glucose 6-phosphate dehydrogenase mutants are promising candidates for industrial enzyme preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1This is the vector plasmid map of the TmG6PDH-R7 mutant;

[0025] Figure 2 Schematic diagram of high-throughput screening of a TmG6PDH mutant library with improved specific enzyme activity; yellow-colored mutants in the figure are candidate mutants with improved specific enzyme activity;

[0026] Figure 3 NADP-based + and NMN + Comparison chart of the specific enzyme activity (37°C);

[0027] Figure 4 This is the result of large-volume affinity purification SDS-PAGE; lane M: marker; lane 1: supernatant; lane 2: precipitate; lane 3: concentrated sample after elution; the size of the TmG6PDH mutant is 58 kDa. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described below in conjunction with specific examples. The advantages and features of the present invention will become clearer as the description progresses. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the scope of protection of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or the 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.

[0029] The following materials are used in the embodiments of the present invention:

[0030] D-glucose 6-phosphate (G6P), product of Shanghai Macklin Co., Ltd., product number: D810527-100 mg;

[0031] Oxidized NMN + , Shanghai Aladdin Company product, item number: N131850-25g;

[0032] Escherichia coli BL21 (DE3) competent cells, a product of Shanghai Weidi Company, catalog number: EE1002S;

[0033] pET-28a vector plasmid, product of Jiangsu Saisuofei Biotechnology Co., Ltd., order number: G69045

[0034] Gel recovery / PCR purification kit, OMEGA product, catalog number: D2000-02;

[0035] QuickMutation TM Gene random mutagenesis kit, product of Beijing Biyuntian Company, catalog number: D0219S;

[0036] Deoxyribonucleotide premix (dNTP Mixture), product of Beijing Biyuntian Pharmaceutical Co., Ltd., catalog number: D7371;

[0037] pEASY-Basic Seamless Cloning and Assembly Kit, product of Quanshijin Co., Ltd., catalog number: CU201-02;

[0038] Water-soluble tetrazolium-1 (WST-1), product of Shanghai Macklin Co., Ltd., product number: W820525-250mg;

[0039] Flavin adenine dinucleotide disodium salt hydrate (DI), product of Shanghai Aladdin Co., Ltd., product number: F100614-100mg;

[0040] Dpn I enzyme, product of ThermoFisher, catalog number: ER1701;

[0041] Ni-NTA filler (High Affinity Ni-Charged Resin FF), product of Nanjing GenScript Biotechnology Co., Ltd., catalog number: L00666.

[0042] 1. Vector construction method of TmG6PDH mutant:

[0043] Using QuickMutation TM The random gene mutagenesis kit uses the TmG6PDH-R7 vector pET28a-Ptac-tmg6pdh-R7 or a vector containing the optimal mutant from each round as a template, designs primers, and performs PCR amplification. The TmG6PDH-R7 upstream primer (SEQ ID NO. 12) is 5'-actttaagaaggagatatcatatgaagtgcagtctgggattgg-3', and the TmG6PDH-R7 downstream primer (SEQ ID NO. 13) is 5'-ccggatctcagtggtggtggtggtggtgCTCGAGcag-3'.

[0044] The 25 μL PCR reaction system was as follows: 40 ng of plasmid template DNA, 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 5 μL of 10x EnhancerMut buffer, 5 μL of dNTPs (2.5 mM), 0.4 μL of RandomMut DNA polymerase, and double-distilled water was added to the volume of 25 μL.

[0045] After mixing, the mixture was briefly centrifuged and PCR was performed. PCR amplification conditions were as follows: initial denaturation at 94°C for 3 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 2 minutes, followed by a final incubation at 72°C for 10 minutes. The PCR product was digested with Dpn I for 30 minutes and then purified by gel purification using a PCR product purification kit.

[0046] The purified PCR product was ligated with the linearized vector fragment pET28aPtac- (from the reference Li, Qiangzi, et al. "Stoichiometric Regeneration of Biomimetic Nicotinamide Coenzyme Powered by Biomass Sugars via In Vitro Synthetic Enzymatic Biosystems." ChemSusChem 18.4(2025):e202401263) using the seamless cloning ligation kit, mixed thoroughly, and reacted at 50°C for 15 minutes. The ligation products were transformed into Escherichia coli BL21(DE3) competent cells, plated onto LB solid medium plates containing 50 μg / mL of kanamycin, and cultured overnight.

[0047] 2. High-throughput screening method for plate-based positive clones:

[0048] Wrap the plate that has been transformed and cultured for 24 hours with sealing film and place it in a 60°C water bath for 60 minutes to disrupt the cells, kill the endogenous proteins of E. coli, and degrade the endogenous reduced coenzymes. Pour the color development solution on the plate and develop the color. The reaction system of the color development solution on the plate is: 0.5% w / v agarose, 50mM Tris-HCl (pH 7.4), 0.1% w / v sodium azide, 50μg / mL chloramphenicol, 150μM WST-1, 0.4mg / mL DI (2.8μg / mL), 2mM G6P, 2.5mM NMN + In the reaction system, flavin adenine dinucleotide disodium salt hydrate (DI) oxidizes the reduced NMNH to NMN + , G6PDH converts oxidized NMN +The enzyme is reduced to NMNH, regenerating the reduced biomimetic coenzyme. Allow to stand at room temperature for color development. Yellow colonies are potential positive clones to be selected.

[0049] 3. Expression of TmG6PDH and its mutants:

[0050] Pick the potential positive single clone that shows yellow color, use the plasmid extraction kit to extract the plasmid DNA, and then use it to directly transform the BL21 (DE3) strain. Pick the single clone to expand the culture and extract the plasmid for sequencing. Compare the sequencing results with the template DNA, and select the one with sense mutation and the same plasmid. The sequencing results of two colonies are consistent and the protein expression and purification are carried out in the next step. The BL21 (DE3) strain containing the mutant gene after sequencing is inoculated with the seed liquid in 200mL LB liquid medium at a ratio of 1:100 (v / v), and cultured at 37 ° C, 180 rpm until the OD 600 When the p-value was 0.6-0.8, IPTG was added to induce the mixture at a final concentration of 0.2 mM and cultured overnight at 30°C. The cells were collected by centrifugation at 8000 rpm for 10 min and the crude mutant protein was obtained by ultrasonic disruption. The electrophoretically pure mutant protein was obtained by nickel column purification, ultrafiltration concentration, and buffer exchange.

[0051] 4. Determination of the enzyme activity of TmG6PDH and its mutants:

[0052] Enzyme activity assay reactions were carried out at 37°C in 50 mM Tris-HCl buffer (pH 7.5).

[0053] The 200 μL reaction system includes 5 mM MgCl2, 0.5 mM MnCl2, 1 mM NADP + or 5mM NMN + , 2mM G6P and an appropriate amount of TmG6PDH enzyme. By measuring the absorbance at 340nm, according to the molar absorption coefficient of NADPH and NMNH 6.22mM - 1 cm -1 The amount of NADPH or NMNH generated was calculated, and then the specific enzyme activity was calculated.

[0054] 5. Enzyme kinetic curve determination method of TmG6PDH and its mutants:

[0055] The reaction system for enzyme kinetics curves is the same as that for comparative enzyme activity, except for the difference in substrate concentration. For the NMN reaction system, the NMN concentration range is 0-10mM. For the NADP reaction system, the NADP concentration range is 0-1mM.

[0056] The following will disclose specific embodiments for implementing the present application, as well as corresponding comparative examples to demonstrate the relevant technical effects of the present application.

[0057] Example 1 Obtaining the parent TmG6PDH protease gene

[0058] At present, the glucose 6-phosphate dehydrogenase mutant TmG6PDH-R7 with the highest specific enzyme activity with NMN preference is derived from the reference (Li, Qiangzi, et al. "Stoichiometric Regeneration of Biomimetic Nicotinamide Coenzyme Powered by Biomass Sugars via In Vitro Synthetic Enzymatic Biosystems." ChemSusChem 18.4(2025):e202401263). The gene sequence of the above-mentioned protease mutant (nucleotide sequence as shown in SEQ ID NO.3) is preceded by a sequence InsertSequence (see sequence SEQ ID NO.11), which contains the constitutive Ptac constitutive promoter sequence, the lac operator sequence and the ribosome recognition sequence RBS (see Figure 1 ). Subsequently, a biological company was commissioned to synthesize the above DNA sequence, and then inserted into the commercial vector pET-28a plasmid after the T7 promoter sequence and before the 6xHis tag sequence to obtain the recombinant plasmid pET28a-Ptac-tmg6pdh-R7 (see Figure 1 ) and transformed into the competent Escherichia coli BL21 (DE3) strain.

[0059] Example 2 Construction of NMN-preferring TmG6PDH protease mutant library and screening of high-activity mutants

[0060] (1) Random mutagenesis based on error-prone PCR technology to construct an acidic protease mutant library

[0061] The primer design was the same as the upstream and downstream primers of the parent TmG6PDH-R7, which were derived from the reference (Li, Qiangzi, et al. "Stoichiometric Regeneration of Biomimetic Nicotinamide Coenzyme Powered by Biomass Sugars via In Vitro Synthetic Enzymatic Biosystems." ChemSusChem18.4(2025):e202401263). Error-prone PCR was performed using the parent TmG6PDH-R7 protease genome obtained in Example 1 as a template.

[0062] The amplification reaction system is: DNA template 0.5-5ng / μL, 10μM upstream primer 2μL, 10μM downstream primer 2μL, 10X RandomMut buffer 5μL, 0.25mM dNTP 2μL, 10XMutation enhancer 2μL, RandomMutDNA polymerase 1μL, ddH2O up to 50μL.

[0063] The amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 3 min, 30 cycles; and extension at 72°C for 10 min.

[0064] The PCR amplification product was subjected to 1% agarose gel electrophoresis and recovered using a small DNA recovery kit to obtain the protease gene TmG6PDH-R7-Mx (x represents the number of the randomly mutated gene) carrying the random mutation. The purified product fragment was ligated into the pET28a-Ptac vector to generate the recombinant plasmid pET28a-Ptac-tmg6pdh-R7-Mx. This recombinant plasmid was then transformed into a competent Escherichia coli BL21(DE3) strain, plated, and cultured overnight at 37°C.

[0065] (2) Screening of highly active acidic protease mutants

[0066] Wrap the plate that has been transformed and cultured for 24 hours with sealing film and place it in a 60°C water bath for 60 minutes to disrupt the cells, kill the endogenous proteins of E. coli, and degrade the endogenous reduced coenzymes. Pour the color development solution on the plate and develop the color. The reaction system of the color development solution on the plate is: 0.5% w / v agarose, 50mM Tris-HCl (pH 7.4), 0.1% w / v sodium azide, 50μg / mL chloramphenicol, 150μM WST-1, 0.4mg / mL DI (2.8μg / mL), 2mM G6P, 2.5mM NMN + The colonies that show the most obvious yellow color are potential positive clones to be selected (see Figure 2 ), select 1 to 3 on each board.

[0067] (3) Sequence determination

[0068] The protease gene sequence of the recombinant strain obtained by screening was extracted and sequenced (Hangzhou Qingke Biological Co., Ltd.); the mutant protein was obtained by protein expression and nickel column purification; 1mM NADP + or 5mM NMN + As a coenzyme, the specific enzyme activity of the mutant was determined.

[0069] As the starting protein, the R7 mutant (nucleotide sequence shown in SEQ ID NO. 3) was incubated with 1 mM NADP at 37°C. + and 5mM NMN + The specific enzyme activities were 0.25U / mg and 0.21U / mg respectively.

[0070] After the first round of random mutagenesis and high-throughput screening, mutant L446F was obtained. This mutant was resistant to 1 mM NADP at 37°C. + and 5mM NMN + The specific enzyme activities were 0.17 U / mg and 0.32 U / mg, respectively. The optimal mutant L446F obtained in this round was named TmG6PDH-R8, and its nucleic acid sequence is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.6.

[0071] Using R8 as template, after the second round of random mutagenesis and high-throughput screening, the mutant L123H / I249V / G297E was obtained. This mutant was resistant to 1mM NADP at 37℃. + and 5mM NMN + The specific enzyme activities were 0.21 U / mg and 0.84 U / mg, respectively. The optimal mutant L123H / I249V / G297E obtained in this round was named TmG6PDH-R9, and its nucleic acid sequence is shown in SEQ ID NO.7, and its amino acid sequence is shown in SEQ ID NO.8.

[0072] Using R9 as template, after the third round of random mutagenesis and high-throughput screening, mutant V419A was obtained. This mutant was resistant to 1 mM NADP at 37°C. + and 5mM NMN + The specific enzyme activities were 0.19 U / mg and 1.05 U / mg, respectively. The optimal mutant V419A obtained in this round was named TmG6PDH-R10, and its nucleic acid sequence is shown in SEQ ID NO.9, and its amino acid sequence is shown in SEQ ID NO.10.

[0073] After three rounds of random mutagenesis and high-throughput screening, the optimal mutant R10 showed a 0.76-fold decrease in specific enzyme activity to 1 mM NADP+ at 37°C compared to TmG6PDH-R7 (nucleotide sequence shown in SEQ ID NO.3), and a 0.76-fold decrease in specific enzyme activity to 1 mM NMN at 37°C. + The specific enzyme activity was increased by 7.3 times (see Figure 3 After each round of mutation, the coenzyme preference and kinetic parameters of the optimal mutant are shown in Table 1.

[0074] Table 1 Kinetic parameters of TmG6PDH and its mutants

[0075]

[0076] Sequencing revealed that the TmG6PDH-R10 protease mutant contained five additional mutations compared to the parent protease R7: L446F, L123H, I249V, G297E, and V419A. Furthermore, compared to the wild-type protease TmG6PDH-WT (nucleotide sequence shown in SEQ ID NO. 1, amino acid sequence shown in SEQ ID NO. 2), the TmG6PDH-R10 protease mutant contained 12 additional mutations: G31F, A32Q, T37M, T66L, Y94F, P126G, L129A, L446F, L123H, I249V, G297E, and V419A.

[0077] Example 3 Large-volume preparation of TmG6PDH protease mutants

[0078] The glucose-6-phosphate dehydrogenase mutant TmG6PDH-R10 plasmid obtained in Example 2 was transformed into Escherichia coli BL21 (DE3) for expression. The specific steps are as follows:

[0079] The positive strains screened were cultured on LB solid medium (50 μg / mL kanamycin) until a single colony was grown. The single colony was picked and inoculated into LB liquid medium. The seed solution was cultured in a shaker at 37°C for 12-16 hours to obtain the seed solution. The seed solution was inoculated into 800 mL LB liquid medium at a ratio of 1:100 (v / v) and cultured at 37°C, 180 rpm until the OD 600 is 0.6~0.8,

[0080] IPTG was added to induce the cells at a final concentration of 0.2 mM and the cells were cultured at 30°C overnight. The cells were collected by centrifugation at 8000 rpm for 30 min and then crushed by ultrahigh pressure homogenizer to obtain the crude enzyme solution of mutant protein. The supernatant protein and the precipitated inclusion body protein were separated by high-speed refrigerated centrifugation. The supernatant protein was purified by His-Tag affinity chromatography, centrifuged by ultrafiltration tube and the buffer was replaced. The target protein was concentrated to an appropriate volume and sampled for SDS-PAGE analysis (see Figure 4 ), and the final protein concentration was determined.

[0081] The above embodiments are only used to illustrate the design concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments.

Claims

1. A kind of NMN + A preferential glucose 6-phosphate dehydrogenase mutant, characterized in that The amino acid sequence is shown as SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.

10.

2. A protein having at least 90% homology to the glucose 6-phosphate dehydrogenase mutant according to claim 1.

3. A gene encoding the glucose 6-phosphate dehydrogenase mutant according to claim 1.

4. The gene according to claim 3, characterized in that The nucleotide sequence of the gene is shown as SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.

9.

5. A vector or cell carrying the gene according to claim 3 or 4.

6. A kind of NMN + The method for preparing a preferential glucose 6-phosphate dehydrogenase mutant is characterized in that: The following steps are involved: Using a vector containing the gene encoding the TmG6PDH mutant as a template, random mutagenesis was performed using error-prone PCR technology. A high-throughput screening method for plate-positive clones was used to select one or more clones with the most obvious color development. The plasmid was extracted, and the vector carrying the mutant gene was introduced into Escherichia coli. The recombinant bacteria were cultured and purified to obtain the glucose 6-phosphate dehydrogenase mutant.

7. The method according to claim 6, characterized in that The coding gene of the TmG6PDH mutant is the sequence shown in SEQ ID NO.3, or the sequence shown in SEQ ID NO.11 is added to the 5' end of the sequence shown in SEQ ID NO.3.

Images