A type of NMN + Preferred glucose-6-phosphate dehydrogenase mutants and their preparation methods

Abstract

This invention discloses a method with NMN + A preferred glucose-6-phosphate dehydrogenase mutant and its preparation method were investigated. Using the TmG6PDH-R7 mutant as the starting protein, random mutations were performed using error-prone PCR to construct a random mutant library. A high-throughput screening method for positive clones on plates was used to obtain the clones with the most obvious color development. Recombinant bacteria were cultured and purified to obtain the mutants. After three rounds of screening, NMN with amino acid sequences as shown in SEQ ID NO.6, SEQ ID NO.8, or SEQ ID NO.10 was obtained. + A glucose-6-phosphate dehydrogenase mutant with enhanced preference, achieving NMN + The specific enzyme activity of TmG6PDH coenzyme is increased. The mutant with the amino acid sequence shown in SEQ ID NO.10 has more than 5 times higher specific enzyme activity at 37 degrees compared with the R7 mutant with the highest specific enzyme activity, and has good application prospects in industrial production.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering, and relates to a method containing NMN. + A preferred glucose-6-phosphate dehydrogenase mutant, specifically, one capable of using the biomimetic coenzyme NMN + Glucose-6-phosphate dehydrogenase mutants that act as cofactors in redox reactions and their preparation methods. Background Technology

[0002] With NAD(P) + Redox enzymes, 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) + Composed of a nicotinamide region and a phosphorylated or non-phosphorylated adenosine dinucleotide, the former is responsible for electron transfer, while the latter is responsible for recognition and localization at the enzyme's active site. Biomimetic cofactors primarily use nicotinamide as the core, formed by replacing the adenosine dinucleotide in natural cofactors with more stable structures such as sugars, phenyl groups, or benzyl groups. 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 applications of biocatalytic reactions. However, the catalytic ability of oxidoreductases utilizing biomimetic coenzymes and the regeneration of reduced-state biomimetic coenzymes are problems that urgently need to be solved.

[0004] In in vitro multi-enzyme molecular machines catalyzing the production of hydrogen and bioelectricity from monosaccharides and polysaccharides, coenzymes are crucial for mediating electron and proton transfer and ultimately generating products. This study investigated the biomimetic coenzyme NMN for glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) in cell-free in vitro multi-enzyme systems for hydrogen and electricity production. +Preference modification has significant application value.

[0005] Recent research, through multiple rounds of directed evolution, has significantly improved the utilization of these enzymes by the biomimetic coenzyme NMN. + 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 is 2.8 U mg. -1 (60℃), this is the highest level reported so far, but still far below the level based on NADP. + The specific enzyme activity of wild-type glucose-6-phosphate dehydrogenase (14 Umg) -1 Therefore, based on NMN + In the industrial production of hydrogen and bioelectricity using in vitro multi-enzyme systems, there is an urgent need for further NMN-based NMN synthesis of glucose-6-phosphate dehydrogenase. + Preferred modifications were made to achieve a complete replacement of the natural coenzyme NADPH with the biomimetic coenzyme NMN. Summary of the Invention

[0006] The purpose of this invention is to provide a biomimetic coenzyme NMN. + A preferred glucose-6-phosphate dehydrogenase mutant and its preparation method were obtained. Through extensive mutation screening based on existing mutants, a mutant with significantly enhanced enzyme activity was finally obtained.

[0007] This invention is achieved through the following technical solution:

[0008] The starting protein was TmG6PDH-R7, with the amino acid sequence shown in SEQ ID NO.4. This protein had been thermally stabilized and subjected to primary directed evolution modification. Error-prone PCR was used to randomly mutate this gene, and positive clones were screened using a high-throughput plate cloning method.

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

[0010] (a) First round of modification: Using TmG6PDH-R7 (SEQ ID NO.4) as the starting protein, random mutations were performed using error-prone PCR to construct a glucose-6-phosphate dehydrogenase mutant library. A plate-based high-throughput screening method was used to obtain the NMN-encoding mutant. + The nucleic acid of a peptide of a preference-enhanced glucose-6-phosphate dehydrogenase mutant, the protein sequence of which is shown in SEQ ID NO.6 (R8);

[0011] (b) Second round of modification: Starting with the optimal mutant R8 from the first round, random mutations were performed using error-prone PCR to construct a glucose-6-phosphate dehydrogenase mutant library. A high-throughput screening method using plate-based positive clones was employed to obtain the NMN-encoding mutant. + The nucleic acid of a peptide of a preference-enhanced glucose-6-phosphate dehydrogenase mutant, the protein sequence of which is shown in SEQ ID NO.8 (R9);

[0012] (c) Third round of modification: Starting with the optimal mutant R9 from the second round, random mutations were performed using error-prone PCR to construct a glucose-6-phosphate dehydrogenase mutant library. A high-throughput screening method using plate-based positive clones was employed to obtain the NMN-encoding mutant. + The nucleic acid of a polypeptide of a preference-enhanced glucose-6-phosphate dehydrogenase mutant, the protein sequence of which is shown in SEQ ID NO.10 (R10);

[0013] In summary, after three rounds of modification, NMN was obtained. + A preferred glucose-6-phosphate dehydrogenase mutant, with an amino acid sequence as shown in SEQ ID NO.6, SEQ ID NO.8, or SEQ ID NO.10.

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

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

[0016] In addition to the aforementioned mutants R8, R9, and R10, other mutants possessing NMN... + A preferred glucose-6-phosphate dehydrogenase mutant, whose amino acid sequence has at least 80% homology, and especially at least 90% homology, with the amino acid sequences shown in SEQ ID NO.6, SEQ ID NO.8, and SEQ ID NO.10.

[0017] The present invention also provides a product containing NMN. + The preparation method of the preferred glucose-6-phosphate dehydrogenase mutant is as follows:

[0018] Using a vector containing the coding gene of the TmG6PDH mutant (pET28a-Ptac-tmg6pdh-R7 in this embodiment) as a template, a glucose-6-phosphate dehydrogenase mutant library was constructed by random mutagenesis using error-prone PCR. A plate-based high-throughput screening method was employed to obtain one or more clones with the most pronounced colorimetric activity. Plasmids were then extracted and mutation sites were analyzed by sequencing. The vector carrying the mutant gene was introduced into *E. coli* BL21(DE3), and recombinant bacteria were cultured. Glucose-6-phosphate dehydrogenase mutants were obtained through heat treatment or nickel column purification.

[0019] In some embodiments, 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.

[0020] The nucleotide sequence encoding wild-type glucose-6-phosphate dehydrogenase, as shown in SEQ ID NO.1, is derived from *Thermotoga maritima* and synthesized by codon optimization of the sequence with UniProt accession number Q9X0N9 in *E. 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 this invention are as follows:

[0023] After three rounds of constructing a random mutant library and high-throughput screening, three NMN variants were obtained. + All TmG6PDH mutants with enhanced preference achieved NMN + The specific enzyme activity of TmG6PDH, a coenzyme, is significantly enhanced. In particular, compared to the R7 mutant, which currently has the highest known specific enzyme activity, the screened R10 mutant exhibits a more than 5-fold increase in specific enzyme activity at 37 degrees Celsius. This invention provides a coenzyme with NMN... + Preferred glucose-6-phosphate dehydrogenase mutants are strong candidates for enzyme preparations used in industrial production. Attached Figure Description

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

[0025] Figure 2 This diagram illustrates a high-throughput screening process for a library of TmG6PDH mutants with increased enzyme activity; the yellow areas in the diagram represent candidate mutants with increased enzyme activity.

[0026] Figure 3 TmG6PDH wild-type and mutant based on NADP + and NMN + A comparison chart of enzyme activity (37℃);

[0027] Figure 4 Image of large-volume affinity purification SDS-PAGE results; Lane M: marker; Lane 1: supernatant; Lane 2: precipitate; Lane 3: concentrated sample after elution; TmG6PDH mutant size is 58kDa. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below in conjunction with specific embodiments. The advantages and features of this invention will become clearer with the description. However, it should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the technical solutions of this invention can be made without departing from the spirit and scope of the invention, but such modifications or substitutions all fall within the protection scope of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

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

[0030] D-glucose-6-phosphate (G6P), a product of Shanghai McLean Company, product number: D810527-100mg;

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

[0032] Escherichia coli expression strain BL21(DE3) competent cells, product of Shanghai Weidi Co., Ltd., catalog number: EE1002S;

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

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

[0035] QuickMutation TM Random gene mutation kit, product of Beijing Beyotime Biotechnology Co., Ltd., catalog number: D0219S;

[0036] Deoxyribonucleic acid premix (dNTP Mixture), product of Beijing Beyotime Co., Ltd., item number: D7371;

[0037] pEASY-Basic Seamless Cloning and Assembly Kit, TransGen Biotech product, catalog number: CU201-02;

[0038] Water-soluble tetrazolium-1 (WST-1), a product of Shanghai Maclean Company, catalog number: W820525-250mg;

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

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

[0041] Ni-NTA packing (High Affinity Ni-Charged Resin FF), a product of Nanjing Genscript Co., Ltd., item number: L00666.

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

[0043] Using QuickMutation TM The method of the random mutation kit for genes involves designing primers for PCR amplification using "the vector pET28a-Ptac-tmg6pdh-R7 of TmG6PDH-R7 or a vector containing the optimal mutant for each round" as a template. Specifically, the upstream primer for TmG6PDH-R7 (SEQ ID NO.12) is 5'-actttaagaaggagatatcatatgaagtgcagtctgggattgg-3', and the downstream primer for TmG6PDH-R7 (SEQ ID NO.13) is 5'-ccggatctcagtggtggtggtggtggtgCTCGAGcag-3'.

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

[0045] After mixing, the mixture was briefly centrifuged and then subjected to PCR. The PCR amplification conditions were as follows: pre-denaturation at 94℃ for 3 min; followed by 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 2 min, repeated 30 times, and finally incubated at 72℃ for 10 min. The PCR product was digested with Dpn I enzyme for 30 min and then purified by gel extraction using a PCR product purification kit.

[0046] The purified PCR product was used to ligate with the linear 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 a seamless cloning ligation kit. After mixing, the mixture was incubated at 50°C for 15 min. The ligation products were then transformed into E. coli BL21(DE3) competent cells and plated onto LB agar plates containing 50 μg / mL kanamycin, and cultured overnight.

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

[0048] The transformed plates, cultured for 24 hours, were sealed with sealing film and placed in a 60°C water bath for 60 minutes to lyse the cells, kill endogenous E. coli proteins, and degrade endogenous reduced coenzymes. The colorimetric solution was then poured on for plate development. The reaction system for the upper layer colorimetric solution was as follows: 0.5% w / v agarose, 50 mM Tris-HCl (pH 7.4), 0.1% w / v sodium azide, 50 μg / mL chloramphenicol, 150 μM WST-1, 0.4 mg / mL DI (2.8 μg / mL), 2 mM G6P, 2.5 mM NMN. + In the reaction system, flavin adenine dinucleotide disodium hydrate (DI) oxidizes reduced NMNH to NMN. + G6PDH will oxidize NMN +It is reduced to NMNH, achieving the regeneration of the reduced biomimetic coenzyme. Incubate at room temperature for color development. Colonies showing yellow color are potential positive clones to be picked.

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

[0050] Potentially positive single clones exhibiting a yellow color were selected, and plasmid DNA was extracted using a plasmid extraction kit. This DNA was then directly transformed into the BL21(DE3) strain. Single clones were selected for amplification culture, and plasmids were extracted and sent for sequencing. Sequencing results were compared with the template DNA. Colonies with sense mutations and consistent sequencing results from two colonies of the same plasmid were selected for the next step of protein expression and purification. The BL21(DE3) strain containing the mutant gene was inoculated into 200 mL of LB liquid medium at a seed culture ratio of 1:100 (v / v) and cultured at 37°C and 180 rpm until OD500. 600 The concentration was set at 0.6–0.8, and IPTG was added to a final concentration of 0.2 mM for induction. The cells were then cultured overnight at 30°C. The cells were collected by centrifugation at 8000 rpm for 10 min, and the mutant protein crude enzyme solution was obtained by sonication. The solution was purified by nickel column chromatography, ultrafiltration concentration, and buffer replacement to obtain electrophoretically pure mutant protein.

[0051] 4. Methods for determining the specific enzyme activity of TmG6PDH and its mutants:

[0052] The enzyme activity assay was performed at 37°C in 50 mM Tris-HCl buffer (pH 7.5).

[0053] The 200 μL reaction system included 5 mM MgCl2, 0.5 mM MnCl2, and 1 mM NADP. + Or 5mM NMN + 2 mM G6P and an appropriate amount of TmG6PDH enzyme were added. The absorbance at 340 nm was measured, based on the molar absorptivity of NADPH and NMNH (6.22 mM). - 1 cm -1 Calculate the amount of NADPH or NMNH generated, and then calculate the specific enzyme activity.

[0054] 5. Methods for determining the enzyme kinetic curves of TmG6PDH and its mutants:

[0055] The reaction systems for enzyme kinetic curves are consistent with those for specific enzyme activity reactions, except for differences in substrate concentration. For the NMN reaction system, the NMN concentration range is 0–10 mM. For the NADP reaction system, the NADP concentration range is 0–1 mM.

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

[0057] Example 1: Obtaining the parental TmG6PDH protease gene

[0058] Currently, the glucose-6-phosphate dehydrogenase mutant TmG6PDH-R7 with the highest NMN-preferred specific activity level 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). An InsertSequence (see Sequence SEQ ID NO.11) is added to the beginning of the gene sequence of the above protease mutant (nucleotide sequence as shown in SEQ ID NO.3). This sequence contains, in sequence, the constitutive Ptac promoter sequence, the lac operon sequence, and the ribosome recognition sequence RBS (see...). Figure 1 Subsequently, a biotechnology company was commissioned to synthesize the aforementioned DNA sequence, which was then inserted into the commercial vector pET-28a plasmid after the T7 promoter sequence and before the 6xHis tag sequence, resulting in the recombinant plasmid pET28a-Ptac-tmg6pdh-R7 (see...). Figure 1 The cells were then transformed into competent Escherichia coli BL21(DE3) strains.

[0059] Example 2: Construction of an NMN-biased TmG6PDH protease mutant library and screening for high-activity mutants

[0060] (1) Construct an acidic protease mutant library by performing random mutations based on error-prone PCR technology.

[0061] Primer design was the same as the upstream and downstream primers of the parental TmG6PDH-R7, 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). The parental TmG6PDH-R7 protease genome obtained in Example 1 was used as a template for error-prone PCR.

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

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

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

[0065] (2) Screening for high-activity acidic protease mutants

[0066] The transformed plates, cultured for 24 hours, were sealed with sealing film and placed in a 60°C water bath for 60 minutes to lyse the cells, kill endogenous E. coli proteins, and degrade endogenous reduced coenzymes. The colorimetric solution was then poured on for plate development. The reaction system for the upper layer colorimetric solution was as follows: 0.5% w / v agarose, 50 mM Tris-HCl (pH 7.4), 0.1% w / v sodium azide, 50 μg / mL chloramphenicol, 150 μM WST-1, 0.4 mg / mL DI (2.8 μg / mL), 2 mM G6P, 2.5 mM NMN. + Incubate at room temperature for color development. The colonies that show the most prominent yellow color are the potential positive clones to be picked (see...). Figure 2 Select 1 to 3 items from each board.

[0067] (3) Sequence determination

[0068] The protease gene sequence was extracted from the screened recombinant strains and sequenced (Hangzhou Qingke Biotechnology Co., Ltd.); the mutant protein was obtained through protein expression and nickel column purification; and then purified with 1 mM NADP. + Or 5mM NMN + As a coenzyme, the specific enzyme activity of the mutant was measured.

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

[0070] After a first round of random mutagenesis and high-throughput screening, mutant L446F was obtained. This mutant is 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 a template, the mutant L123H / I249V / G297E was obtained after a second round of random mutagenesis and high-throughput screening. This mutant showed resistance to 1 mM NADP at 37 °C. + and 5mM NMN + The specific enzyme activities were 0.21 U / mg and 0.84 U / mg, respectively. The optimal mutant obtained in this round, L123H / I249V / G297E, 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 a template, mutant V419A was obtained after a third round of random mutagenesis and high-throughput screening. This mutant showed resistance 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, compared with TmG6PDH-R7 (nucleotide sequence shown in SEQ ID NO.3), the optimal mutant R10 showed a 0.76-fold decrease in specific activity against 1 mM NADP+ at 37℃, and a decrease in specific activity against 1 mM NMN. + The specific enzyme activity increased by 7.3 times (see...) Figure 3 Table 1 shows the coenzyme preference and kinetic parameters of the optimal mutant after each round of mutation.

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

[0075]

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

[0077] Example 3: Large-volume preparation of the TmG6PDH protease mutant

[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 selected positive strains were cultured on LB solid medium (50 μg / mL kanamycin) until single colonies grew. Single colonies were picked and inoculated into LB liquid medium and cultured in a shaker at 37°C for 12-16 h to obtain a seed culture. The seed culture was then inoculated into 800 mL of LB liquid medium at a ratio of 1:100 (v / v) and cultured at 37°C and 180 rpm until OD reached [the desired growth rate]. 600 It ranges from 0.6 to 0.8.

[0080] Induction was performed with IPTG at a final concentration of 0.2 mM, and the cells were cultured overnight at 30°C. The cells were collected by centrifugation at 8000 rpm for 30 min using a high-speed refrigerated centrifuge. The resulting protein was homogenized using an ultra-high pressure homogenizer to obtain a crude enzyme solution of the mutant protein. High-speed refrigerated centrifugation was used to separate the supernatant protein from the precipitated inclusion body protein. His-Tag affinity chromatography was used to purify the supernatant protein. The protein was then centrifuged using an ultrafiltration tube, and the buffer was replaced. The target protein was concentrated to an appropriate volume, and samples were analyzed by SDS-PAGE (see [link to SDS-PAGE analysis]). Figure 4 ), and determine the final protein concentration.

[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 substance containing NMN + Preferred glucose-6-phosphate dehydrogenase mutants, characterized by, The amino acid sequence is shown in SEQ ID NO.6, SEQ ID NO.8, or SEQ ID NO.

10.

2. The gene encoding the glucose 6-phosphate dehydrogenase mutant of claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.5, SEQ ID NO.7, or SEQ ID NO.

9.

4. A vector or cell carrying the gene described in claim 2 or 3.

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