Glucose dehydrogenase or mutant thereof, and use and method in preparation of nmnh

Abstract

A glucose dehydrogenase or a mutant thereof, and a use and method in preparation of NMNH. The glucose dehydrogenase mutant is obtained by mutation at any one or more of position 39, 51, 75, 93, 157, 195, 208, or 236 of a wild type glucose dehydrogenase amino acid sequence.

Description

Glucose dehydrogenase or its mutant and its use and method in preparing NMNH Technical Field

[0001] The present invention relates to the technical fields of genetic engineering and enzyme engineering, and in particular to glucose dehydrogenase or its mutants and their use and method in preparing NMNH. Background Art

[0002] NAD+ (nicotinamide adenine dinucleotide) plays an important role in human health. As we age, the amount of NAD+ in the body gradually decreases. The reduction of NAD+ affects the ability of cells to produce energy, leading to the occurrence of various diseases during aging, such as hearing and vision loss, cognitive and motor dysfunction, immune deficiency, arthritis caused by autoimmune inflammatory response disorders, metabolic disorders, and cardiovascular disease. Increasing the level of NAD+ in the body plays an important role in maintaining biological health. NAD+ has a large molecular mass and it is difficult for it to enter cells. Appropriate supplementation of NAD+ precursors in the body will help increase the level of NAD+ in the body, thereby reducing the occurrence of diseases.

[0003] Nicotinamide mononucleotide (NMN) and reduced nicotinamide mononucleotide (NMNH) are two NAD+ precursors, and supplementation with these two precursors has been shown to effectively increase the body's NAD+ levels. NMNH is the reduced form of NMN and is a more effective NAD+ enhancer than NMN, primarily due to the following advantages: (1) faster NAD+ enhancement; (2) higher NAD+ concentration; (3) lower doses required to increase NAD+ levels; (4) longer-lasting high NAD+ levels in the body; and (5) a wider range of applicable tissues.

[0004] Currently, there are relatively few technologies for synthesizing NMNH. Chemical synthesis and bio-enzyme catalysis have been reported. The chemical synthesis method uses the reducing agent thiourea dioxide (TDO) to reduce NMN to NMNH, and then purifies it using preparative chromatography. In this method, the reducing agent TDO used is irritating to the skin and mucous membranes and is prone to explosive decomposition under high temperature and impact conditions. At the same time, the cost of preparative chromatography purification technology is relatively high. Compared with chemical methods, bio-enzyme catalysis has the advantage of being green and environmentally friendly. The currently reported enzymatic synthesis technology of NMNH is to use NADH pyrophosphatase to catalyze NADH (reduced nicotinamide adenine dinucleotide) to generate NMNH and AMP. This method uses the substrate NADH, the raw material is expensive, and the mass loss rate reaches 50%. From an economic point of view, the commercial value of this technology is low.

[0005] Therefore, current research on the synthesis of NMNH is insufficient, severely limiting the commercialization of this important substance. Bioenzymatic catalysis relies primarily on efficient functional enzymes. Therefore, identifying suitable dehydrogenases to catalyze the reduction of NMN to NMNH will accelerate the commercialization of NMNH.

[0006] Summary of the Invention

[0007] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a use and method of glucose dehydrogenase in the preparation of NMNH, so as to solve the problems in the prior art.

[0008] To achieve the above-mentioned and other related purposes, the present invention provides the use of any of the following substances in the preparation of NMNH: glucose dehydrogenase, a glucose dehydrogenase homologous protein, a gene encoding glucose dehydrogenase or its homologous protein, a nucleic acid construct comprising the encoding gene, a cell comprising the nucleic acid construct, and a cell having an exogenous glucose dehydrogenase or its homologous protein encoding gene integrated into its genome.

[0009] Preferably, the use is the use in catalyzing NMN to prepare NMNH.

[0010] The present invention also provides a cell for producing glucose dehydrogenase, wherein the cell contains the nucleic acid construct or an exogenous polynucleotide as shown in SEQ ID No. 18 is integrated into the genome.

[0011] The present invention also provides a glucose dehydrogenase mutant, which is obtained by mutation at any one or more positions of the 39th, 51st, 75th, 93rd, 157th, 195th, 208th or 236th position of the amino acid sequence of the wild-type glucose dehydrogenase.

[0012] The amino acid sequence of the wild-type glucose dehydrogenase is shown in SEQ ID NO.20.

[0013] In certain embodiments of the present invention, the glucose dehydrogenase mutant is obtained by a combined mutation at positions 39 and 93, or a combined mutation at positions 39 and 75, or a combined mutation at positions 39 and 157, or a combined mutation at positions 39, 93 and 157, or a combined mutation at positions 39, 93 and 75, or a combined mutation at positions 93 and 208, or a combined mutation at positions 51 and 157, or a combined mutation at positions 93 and 157 in the amino acid sequence of the wild-type glucose dehydrogenase.

[0014] In certain embodiments of the present invention, the glucose dehydrogenase mutant is obtained by performing any one of the following mutations on the wild-type glucose dehydrogenase amino acid sequence shown in SEQ ID NO.20: Y39Q single mutation, Y39N single mutation, Y39Q / A93K combined mutation, Y39Q / I75L combined mutation, Y39N / I75S combined mutation, Y39Q / A93K / H157R combined mutation, Y39N / A93P / H157Q combined mutation.

[0015] The present invention also provides an isolated polynucleotide encoding the aforementioned glucose dehydrogenase mutant.

[0016] The present invention also provides a nucleic acid construct, which comprises the isolated polynucleotide, or the nucleotide sequence of the nucleic acid construct is shown as SEQ ID No.19.

[0017] The present invention also provides a cell, wherein the cell contains the nucleic acid construct or the exogenous isolated polynucleotide is integrated into the genome.

[0018] The present invention also provides a method for preparing the glucose dehydrogenase mutant, comprising the following steps: culturing the cells, inducing expression of the glucose dehydrogenase mutant, and collecting and separating the cells after the induction to obtain an enzyme solution of the glucose dehydrogenase mutant.

[0019] The present invention also provides a method for preparing NMNH, which comprises using glucose dehydrogenase or a mutant thereof to catalyze NMN, thereby obtaining NMNH.

[0020] Preferably, the temperature for catalyzing NMN using glucose dehydrogenase is 25°C-40°C.

[0021] Preferably, the pH value of NMN catalyzed by glucose dehydrogenase is 6-10.

[0022] Preferably, when glucose dehydrogenase is used to catalyze NMN, the initial concentration of NMN is 6 to 60 mM based on the total volume of the catalytic system.

[0023] As described above, the glucose dehydrogenase or its mutant and its use and method in preparing NMNH of the present invention have the following beneficial effects: a precedent for the bioenzymatic synthesis of NMNH has been established; when the reaction temperature is controlled at 30°C, pH 8.0, and the initial NMN concentration is 36mM, the amount of NMNH generated by the glucose dehydrogenase catalytic reaction reaches 2.08mM in 12 hours; the technology for the biological preparation of NMNH is green and environmentally friendly, and no toxic chemical reagents are introduced into the reaction system; the raw material of glucose dehydrogenase is low in price, which is conducive to the industrial development of NMNH. Therefore, it can lay the foundation for the application of bioenzymatic catalysis of NMN to synthesize NMNH. The glucose mutant has high catalytic activity and can efficiently catalyze NMN to synthesize NMNH, which can realize the biological preparation of NMNH; the constructed glucose dehydrogenase mutant is used to catalyze NMN to synthesize NMNH, and the NMNH yield can reach 41.2g / L; the technology of biological preparation of NMNH is green and environmentally friendly, and no toxic chemical reagents are introduced into the reaction system; it is conducive to the industrial development of NMNH; NMNH is an effective precursor of NAD+. Based on the reported efficacy of NAD+, the commercial application of NMNH can effectively improve human health and reduce the occurrence of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG1 shows a liquid chromatogram of the reaction of NMN synthesizing NMNH catalyzed by glucose dehydrogenase of the present invention.

[0025] FIG2 shows a liquid chromatogram of the glucose dehydrogenase mutant of the present invention catalyzing the synthesis of NMN into NMNH after 6 hours of reaction.

[0026] FIG3 shows a mass spectrum of NMNH synthesized by the glucose dehydrogenase mutant of the present invention. DETAILED DESCRIPTION

[0027] The present invention provides the use of any of the following substances in the preparation of NMNH: glucose dehydrogenase, a homologous protein of glucose dehydrogenase, a gene encoding glucose dehydrogenase or its homologous protein, a nucleic acid construct comprising the encoding gene, a cell comprising the nucleic acid construct, and a cell having an exogenous gene encoding glucose dehydrogenase or its homologous protein integrated into its genome.

[0028] Homologous proteins, as used herein, refer to proteins with significant amino acid sequence similarity that perform the same or similar functions in different organisms or the same organism. The similarity is 90% or greater, for example, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater. The homologous protein may be a mutant derived from a wild-type glucose dehydrogenase.

[0029] In certain embodiments of the present invention, the use is for catalyzing NMN to prepare NMNH.

[0030] In certain embodiments of the present invention, the amino acid sequence of the glucose dehydrogenase is as shown in any of SEQ ID NOs. 1, 9 to 17, or a sequence having a sequence identity of 90% or more to any of SEQ ID NOs. 1, 9 to 17, and having the functions of the amino acid sequences defined in SEQ ID NOs. 1, 9 to 17. Furthermore, the sequence may be a sequence obtained by substituting, deleting, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the amino acid sequences shown in SEQ ID NOs. 1, 9 to 17, or a sequence obtained by adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus, and having the functions of a polypeptide fragment of the amino acid sequences shown in SEQ ID NOs. 1, 9 to 17. The amino acid sequence may have a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% with the amino acid sequence shown in SEQ ID No. 1, 9 to 17.

[0031] The nucleotide sequences corresponding to the gene encoding the glucose dehydrogenase or its homologous protein include one or more.

[0032] In a specific embodiment, the nucleotide sequence corresponding to the gene encoding glucose dehydrogenase or a homologous protein thereof is shown in SEQ ID No. 18:

[0033] The "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into target cells or tissues. The nucleic acid construct can be various expression vectors, which include a vector skeleton, i.e., an empty vector, and an expression frame.

[0034] The type of expression vector is not specifically limited. An expression vector refers to a nucleic acid molecule that allows the insertion of exogenous nucleotides without disrupting the vector's ability to replicate and / or integrate in a host cell. An expression vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. An expression vector may also include one or more selectable marker genes and other genetic factors. An expression vector is a vector that contains the necessary regulatory sequences to enable transcription and translation of an inserted gene or genes. The expression vector is selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0035] The prokaryotic expression vector is selected from an Escherichia coli expression vector, a Bacillus subtilis expression vector, or a Streptomyces expression vector. In a preferred embodiment, the prokaryotic expression vector is selected from an Escherichia coli expression vector. Compared with other expression systems, the Escherichia coli expression system has a clear genetic background, a short culture cycle, a high expression level of the target gene, and strong anti-contamination ability. The Escherichia coli expression vector is, for example, a pET expression vector, specifically pET28a or pET32a, which can be stably expressed in Escherichia coli. The expression vector can also be a pCW expression vector, a pUC expression vector, or a pPIC9k expression vector.

[0036] The eukaryotic expression vector is selected from a yeast expression vector, an insect expression vector, or a mammalian expression vector. The mammalian expression vector is selected from a retroviral expression vector, a lentiviral expression vector, an adenoviral expression vector, or an adeno-associated viral expression vector. In a preferred embodiment, the eukaryotic expression vector is selected from a retroviral expression vector, which can be stably expressed in a cell line, such as pMSCV.

[0037] In a specific embodiment, the nucleotide sequence of the nucleic acid construct (i.e., the expression vector encoding the glucose dehydrogenase having an amino acid sequence as shown in SEQ ID No. 13) is shown in SEQ ID No. 19:

[0038] The present invention also provides a nucleic acid construct, the nucleotide sequence of the nucleic acid construct is shown as SEQ ID No.19.

[0039] The present invention also provides a cell for producing glucose dehydrogenase, wherein the cell contains the nucleic acid construct or an exogenous polynucleotide as shown in SEQ ID No. 18 is integrated into the genome.

[0040] The production of glucose dehydrogenase using the cells is an existing technology in the art.

[0041] The present invention provides a glucose dehydrogenase mutant, which is obtained by mutation at any one or more positions of the 39th, 51st, 75th, 93rd, 157th, 195th, 208th or 236th position of the amino acid sequence of the wild-type glucose dehydrogenase.

[0042] In certain preferred embodiments of the present invention, the mutation site is selected from position 39, position 75, position 93, position 157, and position 195. The glucose dehydrogenase mutants formed by mutations at these sites have a higher conversion rate of NMN to NMNH.

[0043] The wild-type glucose dehydrogenase is a glucose dehydrogenase of a microorganism, and the microorganism is selected from Escherichia coli, Streptomyces, Bacillus subtilis, Salmonella typhi, mycobacteria, yeast, mold, etc.

[0044] In certain embodiments of the present invention, the amino acid sequence of the wild-type glucose dehydrogenase is shown in SEQ ID NO.20.

[0045] In certain embodiments of the present invention, the glucose dehydrogenase mutant is obtained by a combined mutation at positions 39 and 93, or a combined mutation at positions 39 and 75, or a combined mutation at positions 39 and 157, or a combined mutation at positions 39, 93 and 157, or a combined mutation at positions 39, 93 and 75, or a combined mutation at positions 93 and 208, or a combined mutation at positions 51 and 157, or a combined mutation at positions 93 and 157 in the amino acid sequence of the wild-type glucose dehydrogenase.

[0046] In certain embodiments of the present invention, the mutation at position 39 is selected from Y39Q or Y39N, that is, the tyrosine at position 39 is mutated to glutamine or asparagine.

[0047] In certain embodiments of the present invention, the mutation at position 51 is selected from E51Q, that is, glutamic acid at position 51 is mutated to glutamine.

[0048] In certain embodiments of the present invention, the mutation at position 75 is selected from I75S or I75L, that is, the isoleucine at position 75 is mutated to serine or leucine.

[0049] In certain embodiments of the present invention, the mutation at position 93 is selected from A93K or A93P, that is, the alanine at position 93 is mutated to lysine or proline.

[0050] In certain embodiments of the present invention, the mutation at position 157 is selected from H157R or H157Q, that is, the histidine at position 157 is mutated to arginine or glutamine.

[0051] In certain embodiments of the present invention, the mutation at position 195 is selected from I195E or I195L, that is, the isoleucine at position 195 is mutated to glutamic acid or leucine.

[0052] In certain embodiments of the present invention, the mutation at position 208 is selected from D208N or D208Y, that is, aspartic acid at position 208 is mutated to asparagine or tyrosine.

[0053] In certain embodiments of the present invention, the mutation at position 236 is selected from A236V, that is, the alanine at position 236 is mutated to valine.

[0054] In certain embodiments of the present invention, the glucose dehydrogenase mutant is obtained by performing any one of the following mutations on the wild-type glucose dehydrogenase amino acid sequence shown in SEQ ID NO.20: Y39Q single mutation, Y39N single mutation, Y39Q / A93K combined mutation, Y39Q / I75L combined mutation, Y39N / I75S combined mutation, Y39Q / A93K / H157R combined mutation, Y39N / A93P / H157Q combined mutation.

[0055] The present invention also provides an isolated polynucleotide encoding the aforementioned glucose dehydrogenase mutant.

[0056] In certain embodiments of the present invention, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 49: aactatcaaagtaat (SEQ ID NO. 49).

[0057] In certain embodiments of the present invention, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NOs. 49-31: aataataagggtctt (SEQ ID NO. 50).

[0058] In certain embodiments of the present invention, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO.51:

[0059] The present invention also provides a nucleic acid construct comprising the isolated polynucleotide.

[0060] The type of expression vector is not specifically limited. An expression vector refers to a nucleic acid molecule that allows the insertion of exogenous nucleotides without disrupting the vector's ability to replicate and / or integrate in a host cell. An expression vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. An expression vector may also include one or more selectable marker genes and other genetic factors. An expression vector is a vector that contains the necessary regulatory sequences to enable transcription and translation of an inserted gene or genes. The expression vector is selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0061] The prokaryotic expression vector is selected from an Escherichia coli expression vector, a Bacillus subtilis expression vector, or a Streptomyces expression vector. In a preferred embodiment, the prokaryotic expression vector is selected from an Escherichia coli expression vector. Compared with other expression systems, the Escherichia coli expression system has a clear genetic background, a short culture cycle, a high expression level of the target gene, and strong anti-contamination ability. The Escherichia coli expression vector is, for example, a pET expression vector, specifically pET28a or pET32a, which can be stably expressed in Escherichia coli. The expression vector can also be a pCW expression vector, a pUC expression vector, or a pPIC9k expression vector.

[0062] The eukaryotic expression vector is selected from a yeast expression vector, an insect expression vector, or a mammalian expression vector. The mammalian expression vector is selected from a retroviral expression vector, a lentiviral expression vector, an adenoviral expression vector, or an adeno-associated viral expression vector. In a preferred embodiment, the eukaryotic expression vector is selected from a retroviral expression vector, which can be stably expressed in a cell line, such as pMSCV.

[0063] The host cell is selected from a eukaryotic host cell or a prokaryotic host cell. The eukaryotic host cell is selected from fungi such as yeast, insects, birds, plants, C.elegans or nematodes or mammalian host cells. A non-limiting example of an insect cell is a Spodoptera frugiperda (Sf) cell. The example of a yeast host cell is Saccharomyces cerevisiae (S.cerevisiae), Kluyveromyces lactis (Kluyveromyces lactis, K.lactis) or Yarrowia lipolytica (Yarrowia lipolytica), Pichia pastoris. The example of a mammalian cell is COS cell, baby hamster kidney cell, mouse L cell, LNCaP cell, Chinese hamster ovary (CHO) cell, human embryonic kidney (HEK) cell, African green monkey cell, CV1 cell, Vero or Hep-2 cell. Examples of prokaryotic host cells include bacterial cells, such as E. coli, Streptomyces, B. subtilis, Salmonella typhi, or Mycobacteria.

[0064] Those skilled in the art can transfect the expression vector into host cells according to methods well known in the art to obtain cells containing the gene encoding the glucose dehydrogenase mutant. For example, the expression vector can be introduced into eukaryotic cells by calcium phosphate coprecipitation, electroporation, microinjection, lipofection, or transfection using a polyamine transfection reagent.

[0065] The present invention also provides a cell, wherein the cell contains the nucleic acid construct or an exogenous polynucleotide as shown in any one of SEQ ID NOs. 49 to 51 is integrated into the genome.

[0066] The cells are bacteria or fungi.

[0067] The present invention also provides a method for preparing the glucose dehydrogenase mutant, comprising the following steps: culturing the cells, inducing expression of the glucose dehydrogenase mutant, and collecting and separating the cells after the induction to obtain an enzyme solution of the glucose dehydrogenase mutant.

[0068] In certain embodiments of the present invention, the cell culture conditions include any one of the following: a culture temperature of 25-32° C., a rotation speed of 150-250 rpm, and an LB culture medium.

[0069] In certain embodiments of the present invention, the expression of the glucose dehydrogenase mutant is induced when the culture system has an OD600 of 0.5-1.0.

[0070] In certain embodiments of the present invention, IPTG is used to induce expression of the glucose dehydrogenase mutant.

[0071] In certain embodiments of the present invention, the final concentration of IPTG is 0.3 to 0.6 mM based on the total volume of the culture system. In certain embodiments of the present invention, the induction time is 14 to 18 hours.

[0072] In certain embodiments of the invention, the bacterial cells are collected by centrifugation.

[0073] In certain embodiments of the present invention, the isolation method is: the collected bacterial cells are disrupted by ultrasonication and centrifuged to obtain the enzyme solution of the glucose dehydrogenase mutant.

[0074] The present invention also provides uses of the glucose dehydrogenase mutant, the isolated polynucleotide, the nucleic acid construct, and the cell in preparing NMNH.

[0075] The use is to catalyze NMN to prepare NMNH.

[0076] The present invention also provides a method for preparing NMNH, which comprises using wild-type glucose dehydrogenase or a glucose dehydrogenase mutant to catalyze NMN, thereby obtaining NMNH.

[0077] In certain embodiments of the present invention, the temperature for catalyzing NMN using the glucose dehydrogenase mutant is 25° C.-40° C. For example, the temperature is 25-30° C., 30-35° C., or 35-40° C.

[0078] In certain embodiments of the present invention, the pH value of using a glucose dehydrogenase mutant to catalyze NMN is 6-10, for example, pH 6-7, 7-7.5, 7.5-8, 8-8.5, 8.5-9, or 9-10.

[0079] When the glucose dehydrogenase mutant provided by the present invention is used for an enzyme-catalyzed reaction, the conversion rate of NMN is high when the reaction temperature is controlled at 25-40° C. and the pH is controlled at 7.5-10.0.

[0080] In certain embodiments of the present invention, when glucose dehydrogenase or a mutant thereof is used to catalyze NMN, the final concentration of glucose dehydrogenase or a mutant thereof is 0.5 mg / mL to 200.0 mg / mL based on the total volume of the catalytic system. The final concentration of the glucose dehydrogenase mutant in the catalytic system is, for example, 0.5-10 mg / mL, 2-30 mg / mL, 5-50 mg / mL, 10-100 mg / mL, or 10-200 mg / mL.

[0081] In certain embodiments of the present invention, when the glucose dehydrogenase mutant is used to catalyze NMN, the initial concentration of NMN is 6-60 mM based on the total volume of the catalytic system. For example, the initial concentration of NMN is 6-10 mM, 10-20 mM, 20-30 mM, 30-35 mM, 35-40 mM, 40-50 mM, or 50-60 mM.

[0082] In certain embodiments of the present invention, the catalytic system for catalyzing NMN using glucose dehydrogenase or a mutant thereof includes: glucose dehydrogenase or a mutant thereof, NMN, glucose, NaCl, and pH buffer.

[0083] In certain embodiments of the present invention, the concentration of glucose in the catalytic system is 0.05-2.0 mol / L, based on the total volume of the catalytic system. The concentration of glucose in the catalytic system is, for example, 0.05-0.1 mol / L, 0.1-0.2 mol / L, 0.2-0.3 mol / L, 0.3-0.4 mol / L, 0.4-0.5 mol / L, 0.5-1.0 mol / L, 1.0-1.5 mol / L, or 1.5-2.0 mol / L.

[0084] In certain embodiments of the present invention, the concentration of NaCl in the catalytic system is 0.1-4.0 mol / L based on the total volume of the catalytic system. The concentration of NaCl in the catalytic system is, for example, 0.1-0.5 mol / L, 0.5-1 mol / L, 1-1.5 mol / L, 1.5-2 mol / L, 2-2.5 mol / L, 2.5-3 mol / L, 3-3.5 mol / L, or 3.5-4 mol / L.

[0085] In certain embodiments of the present invention, the pH buffer is, for example, a Tris-HCl buffer.

[0086] In certain embodiments of the present invention, the method specifically involves using the enzyme solution of the glucose dehydrogenase or its mutant to catalyze NMN to obtain NMNH.

[0087] In certain embodiments of the present invention, the preparation method of the glucose dehydrogenase is as follows: culturing the cells for producing glucose dehydrogenase, inducing the expression of glucose dehydrogenase, collecting the cells after the induction and isolating to obtain glucose dehydrogenase enzyme solution.

[0088] In certain embodiments of the present invention, the cell culture conditions include any one of the following: a culture temperature of 25-32° C., a rotation speed of 150-250 rpm, and an LB culture medium.

[0089] In certain embodiments of the present invention, the expression of glucose dehydrogenase is induced when the culture system reaches an OD600 of 0.5-1.0.

[0090] In certain embodiments of the present invention, IPTG is used to induce glucose dehydrogenase expression. In certain embodiments of the present invention, the final concentration of IPTG is 0.3 to 0.6 mM based on the total volume of the culture system. In certain embodiments of the present invention, the induction time is 14 to 18 hours.

[0091] In certain embodiments of the invention, the bacterial cells are collected by centrifugation.

[0092] In certain embodiments of the present invention, the separation method is: the glucose dehydrogenase enzyme solution is obtained by ultrasonically disrupting the collected bacterial cells and centrifuging them.

[0093] In certain embodiments of the present invention, NMN and / or glucose are supplemented during the catalytic process.

[0094] In certain embodiments of the present invention, when the concentration of NMN in the reaction system is lower than 3 mM, NMN and / or glucose are added once. The number of times NMN and / or glucose are added is 1 to 10 times.

[0095] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0096] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0097] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0098] The technical solution adopted in the embodiment is:

[0099] Screening of dehydrogenases that catalyze the synthesis of NMN into NMNH found that lactate dehydrogenase, glycerol dehydrogenase, alcohol dehydrogenase, phosphate dehydrogenase, butanol dehydrogenase, alanine dehydrogenase, serine dehydrogenase, and glucose dehydrogenase can all catalyze the conversion of NMN. Preferably, glucose dehydrogenases such as BhGDH (SEQ ID NO.9), BstGDH (SEQ ID NO.10), BvaGDH (SEQ ID NO.11), and BsuGDH (SEQ ID NO.13) have higher conversion effects, among which BsuGDH (SEQ ID NO.13) is the best.

[0100] Example 1: Screening for dehydrogenases that catalyze the synthesis of NMN into NMNH

[0101] Genetic Screening:

[0102] Various dehydrogenase genes were screened from the NCBI database. The screened genes were: 1. Glucose dehydrogenase, amino acid sequence such as SEQ ID NO. 1 (GenBank: WP_070081367.1; denoted as BaGDH). 2. Lactate dehydrogenase, amino acid sequence such as SEQ ID NO. 2 (GenBank: AKD31452.1; denoted as BvLDHA). 3. Glycerol dehydrogenase, amino acid sequence such as SEQ ID NO. 3 (GenBank: AKD28668.1; denoted as BvGDE). 4. Alcohol dehydrogenase, amino acid sequence such as SEQ ID NO. 4 (GenBank: WP_132105496.1; denoted as BmGDH). 5. Phosphate dehydrogenase, amino acid sequence such as SEQ ID NO. 5 (GenBank: AKD30882.1; denoted as BaPGDH). 6. Butanol dehydrogenase, amino acid sequence such as SEQ ID NO. 6 (GenBank: WP_156240463.1; denoted as BsYQDH). 7. Alanine dehydrogenase, amino acid sequence such as SEQ ID NO. 7 (GenBank: WP_269386815.1; denoted as BaADH). 8. Serine dehydrogenase, amino acid sequence such as SEQ ID NO. 8 (GenBank: AKD31204.1; denoted as BvHDE).

[0103] Construction of host bacteria expressing various dehydrogenases:

[0104] The coding genes of various dehydrogenases (glucose dehydrogenase, lactate dehydrogenase, glycerol dehydrogenase, alcohol dehydrogenase, phosphate dehydrogenase, butanol dehydrogenase, alanine dehydrogenase, and serine dehydrogenase) screened were commissioned to Jiangsu Saisuofei Biotechnology Co., Ltd. for synthesis, cloned into the plasmid pET28a, and transformed into the BL21 (DE3) host to obtain the corresponding engineered bacteria.

[0105] Induce the expression of various dehydrogenases:

[0106] The genetically engineered bacteria expressing glucose dehydrogenase, lactate dehydrogenase, glycerol dehydrogenase, alcohol dehydrogenase, phosphate dehydrogenase, butanol dehydrogenase, alanine dehydrogenase, and serine dehydrogenase were inoculated into 3 mL tubes of LB medium (LB medium composition: 5 g / L yeast extract, 10 g / L trypsin, 10 g / L sodium chloride, pH 7.0) to prepare seed solutions. The culture temperature was controlled at 30°C and the rotation speed was 200 rpm for 16 hours.

[0107] After the incubation, the seed solution was inoculated into a 50 mL shake flask LB medium. The culture conditions were consistent with those used to prepare the seed solution, with a culture temperature of 30°C and a rotation speed of 200 rpm. The OD600 value was detected during the incubation process. When the OD600 value was within the range of 0.5-1.0, IPTG (final IPTG concentration of 0.5 mM) was added to induce the expression of the recombinant enzymes (various dehydrogenases) for approximately 16 h. After the induction, the cells were collected at a centrifugal force of 12,000 g. At the same time, the collected cells were washed twice with Tris-HCl buffer (pH 7.5, 50 mM), and the resulting cells were stored frozen at -20°C.

[0108] Preparation of various dehydrogenases:

[0109] 1 mL of Tris-HCl buffer (pH 7.5, 50 mM concentration) was added to the above-mentioned frozen bacteria to suspend the cells. The suspended bacteria were disrupted with an ultrasonic disruptor (model SCIENTZ-IID, Ningbo Xinzhi Biotechnology Co., Ltd.). The disruption conditions were: ultrasonic for 3 seconds, stop for 6 seconds, and the total ultrasonic time was 5 minutes. The enzyme solution after disruption was centrifuged at 12,000 g and 4°C for 10 minutes. The supernatant obtained was the enzyme solution containing glucose dehydrogenase, lactate dehydrogenase, glycerol dehydrogenase, alcohol dehydrogenase, phosphate dehydrogenase, butanol dehydrogenase, alanine dehydrogenase, and serine dehydrogenase, which can be used for subsequent enzyme catalysis experiments.

[0110] Screening for dehydrogenases that catalyze the synthesis of NMN into NMNH:

[0111] The crude enzyme solutions of glucose dehydrogenase, lactate dehydrogenase, glycerol dehydrogenase, alcohol dehydrogenase, phosphate dehydrogenase, butanol dehydrogenase, alanine dehydrogenase, and serine dehydrogenase obtained above were subjected to enzyme catalysis experiments to investigate the conversion rate of NMN to NMNH. The enzyme catalytic system consisted of 800 μL of 50 mM Tris-HCl buffer, pH 7.5, 100 μL of 60 mM NMN solution, 50 μL of 20 M NaCl solution, 50 μL of 2 M substrate solution, and 5 mg / mL of enzyme solution. The substrate solution selected different substrates according to the enzyme being tested, such as glucose as a substrate for glucose dehydrogenase, lactate as a substrate for lactate dehydrogenase, glycerol as a substrate for glycerol dehydrogenase, ethanol as a substrate for alcohol dehydrogenase, phosphate as a substrate for phosphate dehydrogenase, n-butanol as a substrate for butanol dehydrogenase, alanine as a substrate for alanine dehydrogenase, and serine as a substrate for serine dehydrogenase. Enzyme-catalyzed reaction conditions: temperature 30°C, shaker speed 50 rpm. The reaction was timed starting with the addition of the substrate NMN and continuing for 12 hours. After completion, a 1 mL sample was collected for HPLC analysis of NMN and NMNH content. The appropriate dehydrogenase was initially determined based on the NMNH content in the reaction solution.

[0112] in conclusion:

[0113] According to the data from the enzyme-catalyzed reactions, glucose dehydrogenase, lactate dehydrogenase, glycerol dehydrogenase, alcohol dehydrogenase, phosphate dehydrogenase, butanol dehydrogenase, alanine dehydrogenase, and serine dehydrogenase all reduced the NMN content in the reaction system after a 12-h reaction. The results are shown in Table 1. Among them, glucose dehydrogenase, with an amino acid sequence such as SEQ ID NO. 1 (GenBank: WP_070081367.1; denoted as: BaGDH), performed better.

[0114] Table 1 Different dehydrogenases catalyze NMN

[0115] Example 2: Screening for glucose dehydrogenase that catalyzes the synthesis of NMN into NMNH

[0116] Homologous genes of glucose dehydrogenase (SEQ ID NO. 1, GenBank: WP_070081367.1) were screened from the NCBI database. The amino acid sequences of the screened glucose dehydrogenase homologous genes are as follows: SEQ ID NO.9 (GenBank: WP_202647102.1; recorded as: BhGDH), SEQ ID NO.10 (GenBank: WP_243572584.1; recorded as: BstGDH), SEQ ID NO.11 (GenBank: WP_100741417.1; recorded as: BvaGDH), SEQ ID NO.12 (GenBank: WP_025284235.1; recorded as: BveGDH), SEQ ID NO.13 (GenBank: WP_003246720.1; recorded as: BsuGDH), SEQ ID NO.14 (GenBank: WP_180358503.1; recorded as: EcGDH), SEQ ID NO.15 (GenBank: WP_135800292.1; recorded as: KoGDH), SEQ ID NO.16 (GenBank: EGN2788592.1; denoted as: SeGDH), SEQ ID NO.17 (GenBank: HCD1253839.1; denoted as: CaGDH). The genes encoding the screened glucose dehydrogenase were commissioned to Jiangsu Saisuofei Biotechnology Co., Ltd. for synthesis and cloned into the plasmid pET28a and transformed into the BL21 (DE3) host. The resulting engineered bacteria were designated BL21-BaGDH; BL21-BhGDH; BL21-BstGDH; BL21-BvaGDH; BL21-BveGDH; BL21-BsuGDH; BL21-EcGDH; BL21-KoGDH; BL21-SeGDH; BL21-CaGDH.

[0117] Induce glucose dehydrogenase expression:

[0118] The genetically engineered strains BL21-BaGDH, BL21-BhGDH, BL21-BstGDH, BL21-BvaGDH, BL21-BveGDH, BL21-BsuGDH, BL21-EcGDH, BL21-KoGDH, BL21-SeGDH, and BL21-CaGDH obtained above were inoculated into 3 mL test tubes of LB medium (LB medium composition: 5 g / L yeast extract, 10 g / L trypsin, 10 g / L sodium chloride, pH 7.0) to prepare seed solutions. The culture temperature was controlled at 30°C and the rotation speed was 200 rpm for 16 hours.

[0119] After the incubation, the seed solution was inoculated into a 50 mL shake flask LB medium. The culture conditions were consistent with those used to prepare the seed solution, with a culture temperature of 30°C and a rotation speed of 200 rpm. The OD600 value was detected during the culture process. When the OD600 value was in the range of 0.5-1.0, IPTG (final IPTG concentration of 0.5 mM) was added to induce the expression of the recombinase. The induction time was about 16 h. After the induction, the cells were collected at a centrifugal force of 12,000 g. At the same time, the collected cells were washed twice with Tris-HCl buffer (pH 7.5, concentration of 50 mM) and the resulting cells were stored frozen at -20°C.

[0120] Preparation of glucose dehydrogenase:

[0121] Add 1 mL of Tris-HCl buffer (pH 7.5, 50 mM) to the frozen cells to suspend them. Disrupt the suspended cells using an ultrasonic disruptor (SCIENTZ-IID, Ningbo Xinzhi Biotechnology Co., Ltd.). The conditions were: 3 seconds on, 6 seconds off, for a total of 5 minutes. The enzyme solution was centrifuged at 12,000 g and 4°C for 10 minutes. The resulting supernatant, containing glucose dehydrogenase, was used for subsequent enzyme-catalyzed experiments.

[0122] Screening for glucose dehydrogenase that catalyzes the synthesis of NMN into NMNH:

[0123] The glucose dehydrogenase solution obtained above was used in an enzymatic experiment to examine the conversion efficiency of NMN to NMNH. The enzyme system consisted of 800 μL of 50 mM Tris-HCl buffer (pH 7.5), 100 μL of 60 mM NMN solution, 50 μL of 2 M glucose solution, 50 μL of 20 M NaCl solution, and 5 mg / mL of enzyme solution. The reaction conditions were: temperature 30°C, shaker speed 50 rpm. The enzymatic reaction was initiated with the addition of the substrate NMN. After the addition of NMN, a 1 mL sample was taken from the reaction flask and the initial NMN concentration in the reaction solution was determined by HPLC (denoted as 0 h). After the reaction was completed after 12 h, a 1 mL sample was taken. The residual NMN and NMNH production at the end of the reaction (12 h) were analyzed by HPLC. The conversion efficiency was calculated based on the initial NMN concentration, the residual NMN at the end of the reaction, and the NMNH production.

[0124] The NMNH conversion rate was calculated according to formula ①:

[0125] B(12) / [A(0)–A(12)]×100%①

[0126] Wherein, A(0) is the concentration of NMN at the beginning of the reaction; A(12) is the concentration of NMN in the reaction bottle after 12 h of reaction; B(12) is the concentration of NMNH in the reaction bottle after 12 h of reaction.

[0127] Detection method for NMN and NMNH: A BioCore SAX 5um 4.6*250mm chromatographic column was used, the injection volume was 5μL, the mobile phase was 50mM disodium hydrogen phosphate, the flow rate was controlled at 0.5mL / min, the column oven temperature was 25℃, and the detection wavelength of NMN and NMNH was 225nm.

[0128] The conversion rates of glucose dehydrogenase from different sources after 12 h of reaction are shown in Table 2.

[0129] Table 2 Conversion rate of glucose dehydrogenase from different sources

[0130] Based on the results in Table 2, it was preliminarily determined that glucose dehydrogenases such as BhGDH, BstGDH, BvaGDH, and BsuGDH had high conversion rates, exceeding 10%. In particular, the glucose dehydrogenase BsuGDH achieved a conversion rate of 13.23%. Furthermore, the liquid chromatogram of the glucose dehydrogenase (BsuGDH, SEQ ID NO. 13, GenBank: WP_003246720.1) catalyzing the conversion of NMN to NMNH after 12 hours is shown in Figure 1, demonstrating the production of NMNH.

[0131] Example 3: Optimization of Glucose Dehydrogenase Catalytic Reaction Conditions

[0132] Using strain BL21-BsuGDH as the starting strain, glucose dehydrogenase enzyme solution was prepared after induced expression and cell disruption. The prepared enzyme solution was used to subsequently investigate the catalytic performance of glucose dehydrogenase under different temperature, pH, and initial NMN concentration conditions.

[0133] (1) Catalytic performance of glucose dehydrogenase at different temperatures

[0134] The performance of the prepared glucose dehydrogenase in catalyzing the conversion of NMN to NMNH at different temperatures was investigated. The enzyme catalytic system consisted of 800 μL of 50 mM Tris-HCl buffer (pH 7.5), 100 μL of 60 mM NMN, 50 μL of 2 M glucose, 50 μL of 20 M NaCl, and 5 mg / mL of enzyme solution. The reaction conditions for the enzyme catalysis were as follows: the temperatures were set at 25°C, 30°C, 37°C, and 40°C, and the shaker speed was 50 rpm. The enzyme-catalyzed reaction began with the addition of the substrate NMN and lasted for 12 hours. After completion, a 1 mL sample was removed from the reaction flask, and the amount of NMNH produced in the reaction solution was determined by HPLC. The results of glucose dehydrogenase catalyzing the conversion of NMN to NMNH at different temperatures are shown in Table 3.

[0135] Table 3 The yield of NMNH synthesized by glucose dehydrogenase at different temperatures

[0136] According to the yield of NMNH synthesized by glucose dehydrogenase at different temperatures in Table 3, glucose dehydrogenase can catalyze NMN to synthesize NMNH in the range of 25°C to 40°C. Among them, the amount of NMNH synthesized at 30°C is the highest, reaching 0.794mM.

[0137] (2) Catalytic performance of glucose dehydrogenase at different pH

[0138] The performance of the prepared glucose dehydrogenase in catalyzing the synthesis of NMNH from NMN at different pH values ​​was investigated. The enzyme catalytic system consisted of 100 μL of 60 mM NMN, 50 μL of 2 M glucose, 50 μL of 20 M NaCl, 5 mg / mL enzyme solution, and 800 μL of buffer. The reaction was controlled at different pH values ​​by using different buffers: pH 7.5 (50 mM Tris-HCl buffer at pH 7.5), pH 8.0 (50 mM Tris-HCl buffer at pH 8.0), pH 8.5 (50 mM Tris-HCl buffer at pH 8.5), and pH 9.0 (50 mM Tris-HCl buffer at pH 9.0). The enzymatic reaction conditions were: temperature 30°C, shaker speed 50 rpm. The enzymatic reaction began with the addition of the substrate NMN and continued for 12 hours. After completion, a 1 mL sample was removed from the reaction flask and the amount of NMNH produced in the reaction solution was determined by HPLC. The results of glucose dehydrogenase catalyzing NMN to NMNH at different pH values ​​are shown in Table 4.

[0139] Table 4 The yield of NMNH synthesized by glucose dehydrogenase at different pH values

[0140] According to the yield of NMNH synthesized by glucose dehydrogenase at different pH values ​​in Table 4, glucose dehydrogenase can synthesize NMNH in the pH range of 7.5-9.0, among which the amount of NMNH synthesized at pH 8.0 is the highest, reaching 0.805 mM.

[0141] (3) NMNH production by glucose dehydrogenase at different initial NMN concentrations

[0142] The yield of NMNH synthesized by the prepared glucose dehydrogenase at different initial NMN concentrations was investigated. The enzyme-catalyzed reaction system consisted of 100 μL of NMN stock solution (stock solution concentrations were 60 mM, 180 mM, 360 mM, and 540 mM, respectively), 50 μL of 2 M glucose, 50 μL of 20 M NaCl, and 800 μL of 50 mM Tris-HCl buffer, pH 8.0. The enzyme-catalyzed reaction conditions were: temperature 30°C, shaker 50 rpm. The enzyme-catalyzed reaction began with the addition of the substrate NMN and lasted for 12 h. After completion, a 1 mL sample was removed from the reaction flask, and the amount of NMNH produced in the reaction solution was determined by HPLC. The results of NMNH synthesis by glucose dehydrogenase at different initial NMN concentrations are shown in Table 4.

[0143] Table 5 The yield of NMNH synthesized by glucose dehydrogenase at different initial NMN concentrations

[0144] According to the yield of NMNH synthesized by glucose dehydrogenase at different initial concentrations of NMN in Table 5, it can be seen that glucose dehydrogenase can convert and synthesize NMNH at different concentrations. Among them, when the initial NMN concentration is 36mM, the yield of synthesized NMNH is the highest, reaching 2.08mM.

[0145] Example 4: Screening of mutants for efficient synthesis of NMNH glucose dehydrogenase

[0146] Construction of glucose dehydrogenase and mutants:

[0147] Using glucose dehydrogenase (amino acid sequence shown in SEQ ID NO. 20, GenBank: WP_003246720.1; denoted as BsuGDH) as the starting protein, mutations of BsuGDH were designed through computer simulation. Potentially significant mutation sites were predicted to be single point mutations or pairwise combinations of eight sites: Y39, E51, I75, A93, H157, I195, D208, and A236. DNA sequences encoding the glucose dehydrogenase mutations were amplified by PCR using the constructed recombinant plasmid pET28-BsuGDH (nucleotide sequence shown in SEQ ID NO. 52) and corresponding synthetic primers (synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd.). The PCR products were gel-purified and then fused using a seamless cloning kit (Shanghai Biyuntian Biotechnology Co., Ltd., Cat No. D7010M) to construct the mutant expression plasmid. If there is a single mutation, PCR amplification is performed using the corresponding primers with the recombinant plasmid pET28-BsuGDH as a template, and then fusion is performed to construct an expression plasmid for the mutation. If there are two mutations, a second round of PCR and ligation is performed on the constructed plasmid for the first mutation, using primers for the second mutation, to construct a double-mutation expression plasmid. If there are three mutations, a third round of PCR and ligation is performed on the constructed plasmid for the third mutation, using primers for the third mutation, to construct a triple-mutation expression plasmid.

[0148] in:

[0149] Primer for mutation at position 39 (Y39Q)

[0150] Forward primer (SEQ ID NO. 21): 5'-atcaactatcaaagtaataaac-3'

[0151] Reverse primer (SEQ ID NO. 22): 5'-actttgatagttgataaccacttttg-3'

[0152] Primer for mutation at position 39 (Y39N)

[0153] Forward primer (SEQ ID NO. 23): 5'-gtggttatcaactataatagtaataaac-3'

[0154] Reverse primer (SEQ ID NO. 24): 5'-ttatagttgataaccacttttgcctg-3'

[0155] Primer for mutation at position 51 (E51Q)

[0156] Forward primer (SEQ ID NO. 25): 5'-gtaaaagaacaggtcatcaaggc-3'

[0157] Reverse primer (SEQ ID NO. 26): 5'-gacctgttcttttacctcgttcgga-3'

[0158] Primer for mutation at position 75 (I75S)

[0159] Forward primer (SEQ ID NO. 27): 5'-atgtaaaaaattccgtgcaaacggcaat-3'

[0160] Reverse primer (SEQ ID NO. 28): 5'-cggaattttttacatcttcctctttcg-3'

[0161] Primer for mutation at position 75 (I75L)

[0162] Forward primer (SEQ ID NO. 29): 5'-tctcgtgcaaacggcaattaaggag-3'

[0163] Reverse primer (SEQ ID NO. 30): 5'-tgcacgacattttttacatcttcctc-3'

[0164] Primer for mutation at position 93 (A93K)

[0165] Forward primer (SEQ ID NO.31): 5'-gattaataataagggtcttgaaaatcc-3'

[0166] Reverse primer (SEQ ID NO.32): 5'-cccttattattaatcataatatcgag-3'

[0167] Primer for mutation at position 93 (A93P)

[0168] Forward primer (SEQ ID NO. 33): 5'-gattaataatcccggtcttgaaaatcctg-3'

[0169] Reverse primer (SEQ ID NO. 34): 5'-gaccgggattattaatcataatatcg-3'

[0170] Primer for mutation of site 157 (H157R)

[0171] Forward primer (SEQ ID NO.35): 5'-gtccgctatgcggcaagtaaaggc-3'

[0172] Reverse primer (SEQ ID NO. 36): 5'-tgccgcatagcggacaaataacggc-3'

[0173] Primer for mutation of site 157 (H157Q)

[0174] Forward primer (SEQ ID NO.37): 5'-gtccaatatgcggcaagtaaaggcgg-3'

[0175] Reverse primer (SEQ ID NO.38): 5'-tgccgcatattggacaaataacggc-3'

[0176] Primer for mutation at position 195 (I195E)

[0177] Forward primer (SEQ ID NO.39): 5'-cgccagagaatgctgaaaaattcgc-3'

[0178] Reverse primer (SEQ ID NO.40): 5'-cagcattctctggcgtgttgatcgc-3'

[0179] Primer for mutation at position 195 (I195L)

[0180] Forward primer (SEQ ID NO.41): 5'-gatcaacacgccactcaatgctg-3'

[0181] Reverse primer (SEQ ID NO.42): 5'-agtggcgtgttgatcgcacctg-3'

[0182] 208 site mutation primer (D208N)

[0183] Forward primer (SEQ ID NO.43): 5'-cagaaagctaatgtagaaagcatgattc-3'

[0184] Reverse primer (SEQ ID NO.44): 5'-tacattagctttctgtttagggtcag-3'

[0185] 208 site mutation primer (D208Y)

[0186] Forward primer (SEQ ID NO.45): 5'-cagaaagcttatgtagaaagcatgattc-3'

[0187] Reverse primer (SEQ ID NO.46): 5'-ttctacataagctttctgtttagggtc-3'

[0188] Primer for mutation at position 236 (A236V)

[0189] Forward primer (SEQ ID NO.47): 5'-gaaggaagtcagctacgtcacaggcatc-3'

[0190] Reverse primer (SEQ ID NO.48): 5'-tagctgacttccttcgaagcaagccag-3'

[0191] Nucleotide sequence of pET28-BsuGDH:

[0192] The plasmid encoding the glucose dehydrogenase mutant constructed above was transformed into a BL21 (DE3) host to obtain the corresponding engineered strain.

[0193] Inducible expression of glucose dehydrogenase and its mutants:

[0194] The engineered strains carrying the genes encoding glucose dehydrogenase BsuGDH and the mutants were inoculated into 3 mL of LB tube culture medium, cultured at 30°C and 200 rpm for 16 hours, and the resulting culture was seed liquid. The seed liquid was then inoculated into a 50 mL shake flask of LB medium under the same culture conditions as those used for seed preparation, with a temperature of 30°C and a rotation speed of 200 rpm. The OD600 value was measured during the culture process. When the OD600 value was in the range of 0.5-1.0, IPTG (final IPTG concentration of 0.5 mM) was added to induce expression of the glucose dehydrogenase mutants for approximately 16 hours. After induction, the cells were collected at a centrifugal force of 12,000 g. The collected cells were washed twice with Tris-HCl buffer (pH 7.5, 50 mM) and stored frozen at -20°C.

[0195] Preparation of glucose dehydrogenase and its mutants:

[0196] 1 mL of Tris-HCl buffer (pH 7.5, 50 mM) was added to the frozen cells to suspend the cells. The suspended cells were disrupted using an ultrasonic disruptor (model SCIENTZ-IID, Ningbo Xinzhi Biotechnology Co., Ltd.). The disruption conditions were: ultrasonication for 3 seconds, pause for 6 seconds, and a total of 5 minutes of ultrasonication. The disrupted enzyme solution was centrifuged at 12,000 g and 4°C for 10 minutes. The resulting supernatant was the enzyme solution containing glucose dehydrogenase and mutants, which was used for subsequent enzyme catalysis experiments.

[0197] Performance of glucose dehydrogenase and its mutants:

[0198] The glucose dehydrogenase and mutants obtained above were subjected to enzyme catalysis experiments. The enzyme catalytic system consisted of 800 μL of 50 mM Tris-HCl buffer (pH 7.5), 100 μL of 60 mM NMN solution, 50 μL of 2 M glucose solution, 50 μL of 20 M NaCl solution, and 1 mg / mL of glucose dehydrogenase (calculated based on the total volume of the enzyme catalytic system). The enzyme catalytic reaction conditions were: temperature 30°C, shaker speed 50 rpm. The enzyme catalytic reaction was initiated with the addition of the substrate NMN. After the addition of NMN, a 1 mL sample was taken from the reaction flask and the initial NMN concentration was determined by HPLC (denoted as 0 h). After 6 h of reaction, a 1 mL sample was also taken. The residual NMN content in the reaction solution and the amount of NMNH produced at the end of the reaction (6 h) were determined by HPLC. The conversion rate was calculated based on the initial NMN concentration, the residual NMN content at the end of the reaction, and the amount of NMNH produced.

[0199] The NMNH conversion rate was calculated according to formula ①: B(6) / [A(0)–A(6)]×100%①

[0200] Wherein, A(0) is the concentration of NMN at the beginning of the reaction; A(6) is the concentration of NMN in the reaction bottle after 6 h of reaction; B(6) is the concentration of NMNH in the reaction bottle after 6 h of reaction.

[0201] Detection method for NMN and NMNH: A BioCore SAX 5um 4.6*250mm chromatographic column was used, the injection volume was 5μL, the mobile phase was 50mM disodium hydrogen phosphate, the flow rate was controlled at 0.5mL / min, the column oven temperature was 25℃, and the detection wavelength of NMN and NMNH was 225nm.

[0202] After 6 h of reaction, the concentration and conversion rate of NMN to NMNH catalyzed by glucose dehydrogenase and its mutant are shown in Table 6.

[0203] Table 6 Concentration and conversion rate of NMNH synthesized by glucose dehydrogenase and mutants

[0204] According to the conversion rates of different glucose dehydrogenase mutants in Table 6, Y39 (Y39Q, Y39N, Y39Q / A93K, Y39Q / I75L, Y39N / I75S, Y39Q / A93K / H157R, Y39N / A93P / H157Q), I75 (I75S, I75L, Y39Q / I75L, Y39N / I75S), A93 (A93K, A93P, Y39Q / After mutations at sites such as A93K, Y39Q / A93K / H157R, Y39N / A93P / H157Q), H157 (H157R, H157Q, Y39Q / A93K / H157R, Y39N / A93P / H157Q), and I195 (I195E), the conversion rate of glucose dehydrogenase catalyzing NMN to NMNH is high, and all have commercial prospects for catalyzing NMN to NMNH.

[0205] Among them, the glucose dehydrogenase mutant (BsuGDH Y39Q / A93K / H157R ) catalyzed the synthesis of NMN into NMNH after 6 h of reaction as shown in Figure 3.

[0206] At the same time, mass spectrometry analysis is performed on the NMNH detected in the liquid chromatogram. When the sample is subjected to liquid chromatography detection, it is added to the injection volume, such as the injection volume of 50 μL, and then after the NMNH peak is detected in the liquid chromatogram, a 1.5 mL centrifuge tube is used to collect the liquid-phase separated NMNH sample in the subsequent outflow pipeline. The collected NMNH sample is subjected to mass spectrometry analysis to determine the molecular weight of the synthesized NMNH. Glucose dehydrogenase mutant (BsuGDH Y39Q / A93K / H157R ) catalyzed synthesis of NMNH mass spectrum is shown in Figure 3. As can be seen from the mass spectrum, the molecular weight of the synthesized NMNH is consistent with.

[0207] Example 5: Optimization of enzyme-catalyzed reaction conditions of glucose dehydrogenase mutants

[0208] For several mutation sites such as Y39, I75, A93, H157, and I195 that efficiently synthesize NMNH, one of the combination sites was randomly selected for experiments to optimize the enzyme-catalyzed synthesis of NMNH.

[0209] strain BL21-BsuGDH Y39Q / A93K / H157R The starting strain was a glucose dehydrogenase mutant carrying mutations at the Y39Q, A93K, and H157R sites. After induced expression and cell disruption, a glucose dehydrogenase mutant enzyme solution was prepared. This enzyme solution was used to subsequently investigate the catalytic performance of the glucose dehydrogenase mutant under various conditions, including temperature, pH, and initial NMN concentrations.

[0210] (4) Catalytic performance of glucose dehydrogenase mutants at different temperatures

[0211] The performance of the prepared glucose dehydrogenase mutant in catalyzing NMN to synthesize NMNH at different temperatures was investigated. The enzyme catalytic system consists of: 800 μL of 50 mM pH 7.5 Tris-HCL buffer, 100 μL of 60 mM NMN, 50 μL of 2 M glucose, 50 μL of 20 M NaCl, and 1 mg / ml of glucose dehydrogenase (calculated based on the total volume of the enzyme catalytic system). Enzyme-catalyzed reaction conditions: the temperatures were set at 25°C, 30°C, 37°C, and 40°C, and the shaker speed was 50 rpm. The enzyme-catalyzed reaction started with the addition of substrate NMN and ended after 6 hours. After the end, 1 mL of sample was taken out of the reaction flask, and the amount of NMNH generated in the reaction solution was detected by HPLC. Glucose dehydrogenase mutant BsuGDH Y39Q / A93K / H157R The results of catalyzing NMN to NMNH at different temperatures are shown in Table 7.

[0212] Table 7 Concentration and conversion rate of NMNH synthesized by glucose dehydrogenase mutants at different temperatures

[0213] According to the conversion rates of glucose dehydrogenase mutants in synthesizing NMNH at different temperatures in Table 7, the glucose dehydrogenase mutants can efficiently synthesize NMNH in the range of 25°C to 40°C.

[0214] (5) Catalytic performance of glucose dehydrogenase mutant enzymes at different pH

[0215] The performance of the prepared glucose dehydrogenase mutant in catalyzing the synthesis of NMNH from NMN at different pH values ​​was investigated. The enzyme catalytic system consisted of 100 μL of 60 mM NMN, 50 μL of 2 M glucose, 50 μL of 20 M NaCl, 800 μL of buffer, and 1 mg / mL of glucose dehydrogenase (based on the total volume of the enzyme catalytic system). Different pH values ​​were controlled by using buffers with different pH values: pH 7.5 (50 mM Tris-HCl buffer, pH 7.5), pH 8.0 (50 mM Tris-HCl buffer, pH 8.0), pH 8.5 (50 mM Tris-HCl buffer, pH 8.5), and pH 9.0 (50 mM Tris-HCl buffer, pH 9.0). The enzymatic reaction conditions were: temperature 30°C, shaker speed 50 rpm. The enzymatic reaction began with the addition of the substrate NMN and lasted for 6 h. After completion, a 1 mL sample was removed from the reaction flask and the amount of NMNH produced in the reaction solution was determined by HPLC. Glucose dehydrogenase mutant BsuGDH Y39Q / A93K / H157R The results of catalyzing NMN to NMNH at different pH values ​​are shown in Table 8.

[0216] Table 8 Concentration and conversion rate of NMNH synthesized by glucose dehydrogenase mutants at different pH

[0217] According to the conversion rates of glucose dehydrogenase mutants in synthesizing NMNH at different pH values ​​in Table 8, the glucose dehydrogenase mutants can efficiently synthesize NMNH within the pH range of 7.5-10.0.

[0218] (6) NMNH production by glucose dehydrogenase mutants at different initial NMN concentrations

[0219] The yield of NMNH synthesized by the prepared glucose dehydrogenase mutant at different initial NMN concentrations was investigated. The composition of the enzyme-catalyzed reaction system: 100 μL of NMN mother liquor (mother liquor concentrations were 60 mM, 180 mM, 360 mM, and 540 mM, respectively), 50 μL of 2 M glucose, 50 μL of 20 M NaCl, 800 μL of 50 mM pH 8.0 Tris-HCL buffer, and 1 mg / ml of glucose dehydrogenase (calculated based on the total volume of the enzyme-catalyzed system). Enzyme-catalyzed reaction conditions: temperature 35 ° C, shaker 50 rpm. The enzyme-catalyzed reaction started with the addition of substrate NMN and ended after 12 hours. After the end, 1 mL of sample was taken out from the reaction flask, and the amount of NMNH generated in the reaction solution was detected by HPLC. Glucose dehydrogenase mutant BsuGDH Y39Q / A93K / H157R The results of synthesizing NMNH at different initial NMN concentrations are shown in Table 9.

[0220] Table 9 Concentration and conversion rate of NMNH synthesized by glucose dehydrogenase mutants at different initial NMN concentrations

[0221] According to the conversion rate of glucose dehydrogenase mutants in converting NMN with different initial concentrations into NMNH in Table 9, it can be seen that the glucose dehydrogenase mutants can efficiently synthesize NMNH at different concentrations. Among them, when the initial NMN concentration is 6 mM, the conversion rate of synthesizing NMNH is the highest, reaching 92.32%.

[0222] Example 6: Glucose dehydrogenase mutant enzyme produces high NMNH

[0223] Targeting the mutation sites Y39, I75, A93, H157, and I195 for efficient synthesis of NMNH, the Y39Q mutant of glucose dehydrogenase was selected to efficiently synthesize NMNH.

[0224] strain BL21-BsuGDH Y39Q / A93K / H157RThe starting strain (a glucose dehydrogenase mutant carrying mutations at the Y39Q, A93K, and H157R sites) was used for expression induction and cell disruption to prepare a glucose dehydrogenase mutant enzyme solution. The prepared enzyme solution was used to catalyze the synthesis of NMN into NMNH.

[0225] The enzyme-catalyzed reaction system consists of: 100 μL of 360 mM NMN mother solution, 50 μL of 2 M glucose, 50 μL of 20 M NaCl, 800 μL of 50 mM pH 8.0 Tris-HCL buffer, and 1 mg / ml of glucose dehydrogenase (calculated based on the total volume of the enzyme-catalyzed system). Enzyme-catalyzed reaction conditions: temperature 35°C, shaker 50 rpm. The enzyme-catalyzed reaction starts with the addition of substrate NMN. When the concentration of NMN in the reaction solution is lower than 3 mM, 100 μL of 360 mM NMN mother solution and 50 μL of 2 M glucose are added to the reaction system. After adding 4 times, the reaction is terminated. After the end, 1 mL of sample is taken out from the reaction flask, and the amount of NMNH generated in the reaction solution is detected by HPLC. From the results of the liquid phase, it was determined that the glucose dehydrogenase mutant BsuGDH Y39Q / A93K / H157R The yield of NMNH catalyzed by NMN synthesis reached 41.2 g / L.

[0226] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of any of the following substances in the preparation of NMNH: glucose dehydrogenase, a homologous protein of glucose dehydrogenase, a gene encoding glucose dehydrogenase or a homologous protein thereof, a nucleic acid construct comprising the encoding gene, a cell comprising the nucleic acid construct, a cell having an exogenous gene encoding glucose dehydrogenase or a variant thereof integrated into its genome.

2. The use according to claim 1, characterized in that The use is to catalyze NMN to prepare NMNH.

3. The use according to claim 1, characterized in that The amino acid sequence of the glucose dehydrogenase is as shown in any of SEQ ID NO.1, 9 to 17, or a sequence having a sequence identity of more than 90% with the amino acid sequence shown in any of SEQ ID NO.1, 9 to 17, and having the functions of the amino acid sequence defined by SEQ ID NO.1, 9 to 17.

4. The use according to claim 1, characterized in that The homologous protein of glucose dehydrogenase is obtained by mutation at any one or more positions of the 39th, 51st, 75th, 93rd, 157th, 195th, 208th or 236th position of the amino acid sequence of wild-type glucose dehydrogenase; preferably, the wild-type glucose dehydrogenase is a glucose dehydrogenase of a microorganism; preferably, the amino acid sequence of the wild-type glucose dehydrogenase is as shown in SEQ ID NO.20; more preferably, the glucose dehydrogenase mutant is obtained by a combined mutation at positions 39 and 93, or a combined mutation at positions 39 and 75, or a combined mutation at positions 39 and 157, or a combined mutation at positions 39, 93 and 157, or a combined mutation at positions 39, 93 and 75, or a combined mutation at positions 93 and 208, or a combined mutation at positions 51 and 157, or a combined mutation at positions 93 and 157 in the amino acid sequence of the wild-type glucose dehydrogenase.

5. The use according to claim 1, characterized in that: The homologous protein of glucose dehydrogenase is a protein obtained by performing any one or more of the following mutations on the basis of the amino acid sequence of wild-type glucose dehydrogenase: 1) the mutation at position 39 is selected from Y39Q or Y39N; 2) the mutation at position 51 is selected from E51Q; 3) the mutation at position 75 is selected from I75S or I75L; 4) the mutation at position 93 is selected from A93K or A93P; 5) the mutation at position 157 is selected from H157R or H157Q; 6) the mutation at position 195 is selected from I195E or I195L; 7) the mutation at position 208 is selected from D208N or D208Y; 8) The mutation at position 236 is selected from A236V.

6. The use according to claim 1, characterized in that: The homologous protein of glucose dehydrogenase is obtained by performing any of the following mutations on the wild-type glucose dehydrogenase amino acid sequence shown in SEQ ID NO.20: Y39Q single mutation, Y39N single mutation, Y39Q / A93K combined mutation, Y39Q / I75L combined mutation, Y39N / I75S combined mutation, Y39Q / A93K / H157R combined mutation, Y39N / A93P / H157Q combined mutation.

7. The use according to claim 1, characterized in that The nucleotide sequence of the gene encoding glucose dehydrogenase is shown as SEQ ID No. 18, or includes the nucleotide sequences shown as SEQ ID NOs. 49-50.

8. The use according to claim 1, characterized in that The nucleic acid construct is an expression vector, and the expression vector is selected from a eukaryotic expression vector or a prokaryotic expression vector; preferably, the prokaryotic expression vector is selected from an Escherichia coli expression vector, a Bacillus subtilis expression vector or a Streptomyces expression vector; more preferably, the nucleotide sequence of the nucleic acid construct is shown in SEQ ID No.

19.

9. A cell for producing glucose dehydrogenase, characterized in that An exogenous polynucleotide as shown in SEQ ID No. 18 is integrated into the genome of the cell.

10. A glucose dehydrogenase mutant, characterized in that: The glucose dehydrogenase mutant is obtained by mutation at any one or more positions of the 39th, 51st, 75th, 93rd, 157th, 195th, 208th or 236th position of the amino acid sequence of the wild-type glucose dehydrogenase.

11. The glucose dehydrogenase mutant according to claim 10, characterized in that The wild-type glucose dehydrogenase is a glucose dehydrogenase of a microorganism; preferably, the amino acid sequence of the wild-type glucose dehydrogenase is shown in SEQ ID NO.

20.

12. The glucose dehydrogenase mutant according to claim 10, characterized in that The glucose dehydrogenase mutant is obtained by a combined mutation at positions 39 and 93, or a combined mutation at positions 39 and 75, or a combined mutation at positions 39 and 157, or a combined mutation at positions 39, 93 and 157, or a combined mutation at positions 39, 93 and 75, or a combined mutation at positions 93 and 208, or a combined mutation at positions 51 and 157, or a combined mutation at positions 93 and 157 in the amino acid sequence of the wild-type glucose dehydrogenase.

13. The glucose dehydrogenase mutant according to claim 10, characterized in that Also includes any one or more of the following features: 1) the mutation at position 39 is selected from Y39Q or Y39N; 2) the mutation at position 51 is selected from E51Q; 3) the mutation at position 75 is selected from I75S or I75L; 4) the mutation at position 93 is selected from A93K or A93P; 5) the mutation at position 157 is selected from H157R or H157Q; 6) the mutation at position 195 is selected from I195E or I195L; 7) the mutation at position 208 is selected from D208N or D208Y; 8) The mutation at position 236 is selected from A236V.

14. The glucose dehydrogenase mutant according to claim 10, characterized in that The glucose dehydrogenase mutant is obtained by performing any of the following mutations on the wild-type glucose dehydrogenase amino acid sequence shown in SEQ ID NO.20: Y39Q single mutation, Y39N single mutation, Y39Q / A93K combined mutation, Y39Q / I75L combined mutation, Y39N / I75S combined mutation, Y39Q / A93K / H157R combined mutation, and Y39N / A93P / H157Q combined mutation.

15. An isolated polynucleotide, characterized in that The polynucleotide encodes the glucose dehydrogenase mutant according to any one of claims 10-14.

16. The isolated polynucleotide according to claim 15, characterized in that The polynucleotide comprises the nucleotide sequence shown as SEQ ID NO.49; preferably, the polynucleotide comprises the nucleotide sequence shown as SEQ ID NO.49-50; more preferably, the nucleotide sequence of the polynucleotide is shown as SEQ ID NO.

51.

17. A nucleic acid construct, characterized in that The nucleic acid construct comprises the isolated polynucleotide according to claim 15 or 16, or the nucleotide sequence of the nucleic acid construct is shown as SEQ ID No.

19.

18. A cell, characterized in that The cell contains the nucleic acid construct of claim 17 or the polynucleotide of claim 15 is integrated into its genome.

19. The method for preparing a glucose dehydrogenase mutant according to any one of claims 10 to 14, characterized in that: The method comprises the following steps: culturing the cell according to claim 18, inducing the expression of the glucose dehydrogenase mutant, and after the induction, collecting and crushing the bacteria to obtain the enzyme solution of the glucose dehydrogenase mutant.

20. A method for preparing NMNH, characterized in that: The preparation method comprises using glucose dehydrogenase or the glucose dehydrogenase mutant according to any one of claims 10-14 to catalyze NMN, thereby obtaining NMNH.

21. The preparation method according to claim 20, characterized in that: The preparation method also includes any one or more of the following features: 1) The temperature for catalyzing NMN using glucose dehydrogenase or its mutant is 25°C-40°C; 2) using glucose dehydrogenase or its mutants to catalyze NMN at a pH of 6-10; 3) When glucose dehydrogenase or its mutant is used to catalyze NMN, the final concentration of the glucose dehydrogenase mutant is 0.5 mg / mL to 200.0 mg / mL based on the total volume of the catalytic system; 4) When glucose dehydrogenase or its mutant is used to catalyze NMN, the initial concentration of NMN is 6 to 60 mM based on the total volume of the catalytic system; 5) The catalytic system for catalyzing NMN using glucose dehydrogenase or its mutant includes: glucose dehydrogenase or its mutant, NMN, glucose, NaCl, and pH buffer.

22. The preparation method according to claim 21, characterized in that: The preparation method also includes any one or more of the following features: 1) The catalytic system using glucose dehydrogenase or its mutant to catalyze NMN includes: glucose dehydrogenase or its mutant, NMN, glucose, NaCl, pH buffer, and the concentration of glucose in the catalytic system is based on the total volume of the catalytic system. 0.05~2.0mol / L, and / or, the concentration of NaCl is 0.1~4.0mol / L; 2) Adding NMN and / or glucose during the catalytic process; preferably, the number of times of adding NMN and / or glucose is 1 to 10 times.

23. The preparation method according to claim 20, characterized in that: The method for preparing glucose dehydrogenase comprises the following steps: culturing the cells according to claim 9, inducing the expression of glucose dehydrogenase, and after the induction, collecting the bacteria and separating to obtain the enzyme solution of glucose dehydrogenase.

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