NRK-producing strain, method for producing nrk, and use thereof
By screening and optimizing the fermentation conditions of NRK5-1, the problem of limited NRK sources was solved, and efficient NMN preparation was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN UNIV
- Filing Date
- 2022-07-26
- Publication Date
- 2026-06-26
AI Technical Summary
The limited availability of nicotinamide ribokinase (NRK) in existing technologies leads to low NMN production efficiency via the nicotinamide ribokinase pathway. There is an urgent need to screen and express NRKs with superior enzymatic properties to improve production efficiency.
A novel NRK-producing bacterium, NRK5-1 (Enterobacter kobei 2020T51), was screened from sludge. An efficient expression system was constructed by optimizing fermentation conditions, and a highly active crude NRK enzyme solution was prepared using the optimal fermentation medium and enzyme conversion reaction.
The crude enzyme solution of strain NRK5-1 catalyzed the production of NMN up to 332.23 μmol/L, with a substrate conversion rate of 33.22%, which is 2.3 times that before optimization, significantly improving the production efficiency of NMN.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of engineered microbial technology, and in particular relates to an NRK production microorganism, a method for producing NRK, and its applications. Background Technology
[0002] Nicotinamide mononucleotide (NMN), as a key precursor to the coenzyme nicotinamide adenine dinucleotide (NAD+), plays a vital role in human health. For example, NMN can improve ischemic cardiovascular and cerebrovascular tissue damage, repair oxidation-related bodily dysfunction, and treat metabolic diseases. Human clinical trials have demonstrated that NMN has good safety and can effectively promote bodily functions. Based on its safety and bioactivity, NMN is widely used in food ingredients, pharmaceuticals, health supplements, and skincare products. Therefore, it is necessary to explore economical and efficient NMN production methods to reduce the cost of NMN raw materials.
[0003] Currently, the relatively mature chemical synthesis methods for NMN are prone to chiral issues, and the residue of organic solvents also limits their market application. Enzymatic NMN preparation, on the other hand, offers advantages such as simple reaction, high product activity, and low pollution, representing the future trend in NMN production. Depending on the substrate, the enzymatic synthesis pathways for NMN are mainly divided into two types: the nicotinamide (NAM) pathway and the nicotinamide ribose (NR) pathway. The nicotinamide ribose pathway is catalyzed by a single enzyme, nicotinamide ribokinase (NRK), which catalyzes the synthesis of NMN from the substrate nicotinamide ribose and ATP. While the nicotinamide ribose pathway has some application in the industrial enzymatic preparation of NMN, its application faces bottlenecks: the sources of NRK are limited, and the sources of NRK that have been characterized are relatively rare. There is an urgent need to screen and express NRKs with excellent enzymatic properties to improve the production efficiency of the nicotinamide ribose pathway. Summary of the Invention
[0004] In view of this, the purpose of the present invention is to provide an NRK-producing bacterium that can efficiently synthesize NRK and has high catalytic activity.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0006] This invention provides an NRK production strain, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24913.
[0007] The present invention also provides a method for producing NRK, comprising the following steps: fermenting and culturing the above-mentioned NRK producing bacteria, crushing the obtained fermentation broth, and obtaining a crude enzyme solution containing NRK.
[0008] Preferably, the fermentation medium for the fermentation culture includes 0%-5% carbon source, 1%-3.5% nitrogen source, 0%-1.25% KH2PO4 and 0.01%-0.06% MgSO4·7H2O.
[0009] Preferably, the carbon source includes rhamnose, glucose, fructose, sucrose, galactose, and soluble starch.
[0010] Preferably, the nitrogen source includes beef meal, yeast extract, beef extract, multivalent peptone, and trypsin.
[0011] Preferably, the pH value of the culture medium during fermentation is 6.0-8.0.
[0012] Preferably, the fermentation culture conditions are: 25℃-40℃, shaking culture for 8h-24h.
[0013] Preferably, the oscillation culture is a shaking culture on a shaker, and the rotation speed of the shaker is 150 r / min-220 r / min.
[0014] The present invention also provides an application of the above-mentioned NRK producing bacteria in the preparation of NMN.
[0015] Preferably, the method includes the following steps: fermenting and culturing the above-mentioned NRK producing bacteria, crushing the bacterial solution to obtain crude enzyme solution, and mixing the crude enzyme solution with the mother liquor containing ATP and NR to obtain NMN.
[0016] The beneficial effects of this invention are:
[0017] This invention screened a novel NRK-producing bacterium, NRK5-1 (Enterobacter kobei2020T51), from sludge. Its crude enzyme solution can catalyze the production of 142.5 μmol / L NMN. The fermentation conditions of the screened strain Enterobacter kobei2020T51 were optimized to construct a high-efficiency expression system. The optimal fermentation conditions were: 1% glucose, 3% polyvalent peptone, 0.75% KH2PO4, and 0.03% MgSO4·7H2O, with an initial pH of 7.0. Fermentation was carried out at 40℃ and 200 rpm in a constant-temperature shaker for 16 h. This optimal fermentation condition was suitable for NRK expression and resulted in high enzyme activity. The crude enzyme solution obtained under these optimal conditions yielded an NMN production of 332.23 μmol / L, with a substrate conversion rate of 33.22%, which is 2.3 times that before optimization.
[0018] Preservation Instructions
[0019] The NRK-producing strain NRK5-1 (Enterobacter kobei 2020T51) of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 19, 2022. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the accession number is CGMCC No. 24913. Attached Figure Description
[0020] Figure 1 Figure 1 shows the elution chromatograms of each component in HPLC. Figure 2 shows the elution chromatogram of NMN standard, Figure 3 shows the elution chromatogram of ATP standard, Figure 4 shows the elution chromatogram of NR standard, and Figure 5 shows the elution chromatogram of the mixed sample of NMN, ATP and NR.
[0021] Figure 2 The standard curve of NMN determined by HPLC;
[0022] Figure 3 The yield of NMN produced by the strain;
[0023] Figure 4 The colony morphology is that of NRK5-1;
[0024] Figure 5 The results are from NRK5-1 Gram staining.
[0025] Figure 6 The results are from a scanning electron microscope (SEM) using the NRK5-1.
[0026] Figure 7 The image shows an agarose gel electrophoresis pattern, where M is the DNA Ladder Mix marker and 1 is the NRK5-1 PCR product.
[0027] Figure 8 BLAST results for strain NRK5-1;
[0028] Figure 9 A phylogenetic tree constructed based on the 16S rDNA sequences of strain NRK5-1 and related genera and species;
[0029] Figure 10 The effect of carbon source type on the cell growth and NMN production of Enterobacter kobei 2020T51 is shown, where —□— represents OD. 600 —■— represents NMN production, and 1-7 correspond to rhamnose, glucose, fructose, sucrose, maltose, galactose, and starch, respectively.
[0030] Figure 11The effect of glucose concentration on the cell growth and NMN production of Enterobacter kobei 2020T51 is shown in Figure 1, where —■— represents OD. 600 —●— represents NMN production;
[0031] Figure 12 The effect of nitrogen source type on the cell growth and NMN production of Enterobacter kobei 2020T51 is shown in Figure 1, where —□— represents OD. 600 —■— represents NMN production, and 1-7 correspond to beef powder, yeast extract, beef extract, polyvalent peptone, peptone, urea, and ammonium chloride, respectively.
[0032] Figure 13 The effect of tryptone concentration on the cell growth and NMN production of Enterobacter kobei 2020T51 is shown in the figure, where —■— represents OD. 600 —●— represents NMN production;
[0033] Figure 14 The effect of KH2PO4 concentration on the cell growth and NMN production of Enterobacter kobei 2020T51 is shown in Figure 1, where —■— represents OD200. 600 —●— represents NMN production;
[0034] Figure 15 The effect of MgSO4·7H2O concentration on the cell growth and NMN production of Enterobacter kobei 2020T51 is shown, where —■— represents OD. 600 —●— represents NMN production;
[0035] Figure 16 The effect of initial pH on the cell growth and NMN production of Enterobacter kobei 2020T51 is shown, where —■— represents OD. 600 —●— represents NMN production;
[0036] Figure 17 The effect of shaker speed on the cell growth and NMN production of Enterobacter kobei 2020T51 is shown, where —■— represents OD. 600 —●— represents NMN production;
[0037] Figure 18 The effect of fermentation temperature on the cell growth and NMN production of Enterobacter kobei 2020T51 is shown, where —■— represents OD. 600 , —●— represent NMN production, a is 25℃, b is 30℃, c is 37℃, d is 40℃. Detailed Implementation
[0038] This invention provides an NRK production strain, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24913.
[0039] This invention employs the following screening model: soil collection → enrichment culture in NR-containing medium → selective plate screening of strains → HPLC detection of enzyme activity → streak plating purification of strains → preservation of NRK-producing strains in glycerol tubes. The above-mentioned NRK-producing strain was successfully screened from sludge at the discharge outlet of the Shanghai Shuze Biotechnology Research Institute. 16S rDNA sequencing revealed that the 16S rDNA length of the NRK-producing strain (NRK5-1) of this invention is 1431 bp, and the specific nucleotide sequence is shown in SEQ ID NO.1. Based on BLAST homology alignment and phylogenetic tree analysis, this strain was named Enterobacter kobei2020T51.
[0040] The present invention also provides a method for producing NRK, comprising the following steps: fermenting and culturing the above-mentioned NRK producing bacteria, crushing the obtained fermentation broth, and obtaining a crude enzyme solution containing NRK.
[0041] In this invention, the fermentation medium preferably comprises 0%-5% carbon source, 1%-3.5% nitrogen source, 0%-1.25% KH₂PO₄, and 0.01%-0.06% MgSO₄·7H₂O; more preferably, it comprises 1%-2% carbon source, 2%-3% nitrogen source, 0.5%-1% KH₂PO₄, and 0.02%-0.04% MgSO₄·7H₂O. In this invention, the carbon source preferably includes rhamnose, glucose, fructose, sucrose, galactose, and soluble starch; more preferably, glucose. The nitrogen source preferably includes beef meal, yeast extract, beef extract, polypeptone, and trypsin; more preferably, beef extract or polypeptone. This invention does not specifically limit the source of each component in the above fermentation medium; commercially available products commonly used in the art can be used.
[0042] In this invention, the pH value of the fermentation medium is preferably 6.0-8.0, more preferably 6.5-7.0. The fermentation conditions are preferably 25℃-40℃ with shaking culture for 8-24 hours, more preferably 37℃-40℃ with shaking culture for 14-20 hours. The shaking culture is preferably carried out using a shaker, and the shaking speed is preferably 150 r / min-220 r / min, more preferably 180 r / min-220 r / min.
[0043] The present invention also provides an application of the above-mentioned NRK producing bacteria in the preparation of NMN, preferably comprising the following steps: fermenting and culturing the above-mentioned NRK producing bacteria, crushing the obtained bacterial solution to obtain crude enzyme solution, mixing the crude enzyme solution with a mother liquor containing ATP and NR to obtain NMN.
[0044] In this invention, the fermentation conditions are the same as above and will not be repeated here. The preferred method for the disruption treatment in this invention is ultrasonic disruption, with the preferred ultrasonic disruption conditions being 30% power, 3 seconds of operation, 5 seconds of intermittent operation, and 8 minutes of ultrasonic disruption. In this invention, the mother liquor preferably also contains MgSO4·7H2O, and the mother liquor (enzyme conversion reaction mother liquor) is more preferably composed of a solution of 1 mM ATP, 1 mM NR, and 1 mM MgSO4·7H2O, and PBS buffer (solvent) with a pH of 7.4. In this invention, when the crude enzyme solution is reacted with the mother liquor containing ATP and NR for enzyme conversion, the preferred volume ratio of the crude enzyme solution to the mother liquor is 1:1, the preferred reaction temperature is 37°C, and the preferred reaction time is 15 minutes. After the reaction is completed, it is preferred to terminate the enzyme conversion reaction, preferably by inactivating it in a 95°C metal bath for 1 minute.
[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0046] Example 1
[0047] Samples were collected from sludge at the sewage outlet of the Shanghai Shuze Biotechnology Research Institute. The collected sludge underwent immediate pretreatment. Three clean sites (A, B, and C) were selected from the sludge sample, and 1g of soil was weighed from each site and placed in a 100mL Erlenmeyer flask. 30mL of sterile physiological saline was added, and the flasks were placed in a constant-temperature shaker at 37℃ and 200rpm for 20min. The shaken suspension was then allowed to stand at room temperature until the soil completely precipitated; the supernatant was the enriched sample.
[0048] The supernatant was aspirated and inoculated into enrichment medium (10 mL / 50 mL Erlenmeyer flask) at an inoculation ratio of 10% (v / v), and enriched in a constant temperature shaker at 37℃ and 200 r / min for 12 h.
[0049] The enriched bacterial culture was serially diluted to 10⁻⁶. -4 10 -5 and 10 -6Spread 50 μL of the diluted solution evenly onto agar plates for isolation. After incubating at room temperature for approximately 20 minutes, invert the plates and incubate overnight at 37°C for 12-16 hours. The next day, using a sterile pipette tip, pick single colonies exhibiting different morphology and color from the plates and inoculate them into seed culture medium (20 mL / 50 mL Erlenmeyer flask). Incubate at 37°C and 200 rpm for 12 hours on a shaker. Then, extract 600 μL of the bacterial solution for glycerol preservation. The remaining bacterial solution is used for strain activity testing and screening.
[0050] The selected strains with high activity were streaked onto plates for isolation. Single colonies with good growth were picked and inoculated into seed culture medium (10 mL / 50 mL Erlenmeyer flask). After incubation at 37 °C and 200 rpm for 12 h on a shaker, 600 μL of sterile 50% glycerol and 600 μL of bacterial culture were transferred into a sterile 1.5 mL EP tube, mixed well, and then frozen at -80 °C.
[0051] The specific components and preparation methods of the culture medium used in the above experiments are shown in Table 1.
[0052] Table 1 Culture Media
[0053]
[0054] Example 2
[0055] Strain activity assay
[0056] The level of NMN generated in the enzyme conversion system was used as an indicator for screening NRK-producing bacteria: the higher the level of NMN generated in the conversion system, the stronger the ability of the strain to express NRK enzyme.
[0057] The bacterial culture preserved in glycerol tubes was inoculated into seed culture medium (10 mL / 50 mL Erlenmeyer flask) at a ratio of 10% (v / v) and incubated overnight for 12 h at 37℃ and 200 rpm on a shaker to prepare seed culture. The next day, 1 mL of seed culture was inoculated into fermentation medium (20 mL / 50 mL Erlenmeyer flask) at a ratio of 5% (v / v) and fermented for 12 h at 37℃ and 200 rpm on a shaker. After fermentation, the OD of the bacterial culture was measured using a spectrophotometer. 600 The crude enzyme solution was prepared by ultrasonic treatment.
[0058] Enzymatic conversion reaction
[0059] (1) Mother solution for enzyme conversion reaction: Prepare a solution containing 1mM ATP, 1mM NR and 1mM MgSO4·7H2O, and use PBS buffer with pH 7.4 as the solvent.
[0060] (2) Enzyme conversion reaction method: Take 50 μL of crude enzyme solution and 50 μL of mother solution into 1.5 mL of EP and gently mix by pipetting. Incubate in a 37℃ biochemical incubator for 15 min. After incubation, immediately place the sample in a 95℃ metal bath for 1 min to inactivate and terminate the enzyme conversion reaction.
[0061] HPLC method for the detection of NMN
[0062] ATP, NMN, and NR in the reaction system were separated using a 100% sodium dihydrogen phosphate solution. Methanol and sodium dihydrogen phosphate were used as the mobile phase for gradient elution to remove impurities from the reaction system. The specific elution procedure is shown in Table 2.
[0063] HPLC detection conditions: ChromCore C18 reversed-phase column (5 μM, 4.6 × 250 mm); mobile phase: A = 0.1 mol / L NaH2PO4·2H2O (pH 5.5), B = 100% methanol; column temperature: 25℃; flow rate: 1.0 mL / min; injection volume: 20 μL; UV detection: 254 nm.
[0064] Table 2 HPLC elution program
[0065]
[0066] (1) Sample preparation: Mix 100 μL of enzyme conversion reaction solution with 700 μL of pH 7.4 PBS buffer, filter the mixture through a 0.22 μm microporous membrane into a chromatographic sample vial (the sample volume in the vial should be greater than 500 μL), and then freeze at -20℃. Considering the detection time and instrument wear and tear, perform two parallel experiments for each sample.
[0067] (2) Determination of NMN standard curve by HPLC: 1 mL of NMN standards with concentrations of 8, 10, 15, 20, 25, 50, and 100 μmol / L were prepared, using PBS buffer at pH 7.4 as the solvent. The data were processed using Origin 2019b software, and a standard curve was plotted with NMN concentration on the x-axis and sample peak area on the y-axis.
[0068] The results are as follows Figure 1 and Figure 2 As shown. By Figure 1The retention times of the product NMN and the substrates ATP and NR (three standard samples) were 4.161 min, 6.161 min, and 6.491 min, respectively. After mixing and injecting these three standard samples, the elution times of NMN, ATP, and NR were 4.150 min, 6.212 min, and 6.569 min, respectively. The retention times of the mixed standard samples were basically consistent with the retention times of single-injection samples, indicating that this HPLC detection method has good reproducibility. Figure 2 The results show that the NMN sample has a narrow peak width, good peak shape, and high peak area accuracy; moreover, the retention time interval between the product NMN and the substrates ATP and NR is over 2 minutes, achieving good separation results. Figure 2 It can be seen that when the NMN standard is in the range of 0-100 μmol / L, the standard curve fitted by high performance liquid chromatography shows good linearity, and its R0 is high. 2 The concentration reached 0.9997. The NMN content in the reaction system was then calculated based on the standard curve equation y = 1.0102x - 2.1334.
[0069] The selected strains were analyzed using HPLC to determine the NMN content in the transformation system. Five strains with high enzyme activity were ultimately screened, and their NMN yields were as follows: Figure 3 As shown in the figure, the five strains were named NRK1-3, NRK2-2, NRK2-5, NRK4-4, and NRK5-1, respectively. The crude enzyme solution and whole cells of these five strains were repeatedly subjected to transformation reactions, and the NMN content in the reaction system was detected by HPLC. The results showed that strain NRK5-1 produced the highest NMN yield, reaching 142.5 μmol / L. Furthermore, NMN was only detected in the crude enzyme solution transformation system; no NMN was detected in the whole cell group, indicating that the nicotinamide ribokinase produced by this strain is an intracellular enzyme.
[0070] Example 3
[0071] Strain identification (using NRK5-1 strain obtained from screening in Example 2)
[0072] Morphological identification
[0073] (1) Observation of colony morphology on plate
[0074] Remove the bacterial culture stored in glycerol tubes at -20°C and inoculate it into seed culture medium (10 mL / 50 mL Erlenmeyer flask) at a ratio of 10% (v / v) for 12 hours. The next day, serially dilute the seed culture to 10. -4 10 -5 and 10 -6The culture medium was evenly spread onto agar plates. The plates were then inverted and incubated at 37°C for approximately 14 hours. The colony morphology was observed the following day. Colonies that grew after 14 hours of incubation in the biochemical incubator resembled... Figure 4 As shown: the colonies are round, milky white, and opaque, with a smooth, upward-convex surface, regular edges, and a diameter of less than 2.5 mm.
[0075] (2) Gram staining microscopy
[0076] Smear preparation: Place a drop of sterile ultrapure water on a clean glass slide. Using an inoculation loop, pick up larger colonies grown on the plate and transfer them into the water droplet on the slide, spreading a thin layer of liquid onto the slide. After smearing, place the slide over an alcohol lamp to heat and evaporate the water, thus fixing the sample.
[0077] Staining: Add an appropriate amount of oxalic acid crystal violet stain to the sample area on the slide, and rinse with distilled water after initial staining for 1 min. Mordant with iodine solution for 1 min, rinse with distilled water and air dry. Decolorize with 95% ethanol, allowing the eluent to become colorless before air drying. Counterstain with safranin solution for 1 min, rinse with distilled water, and air dry.
[0078] The strain NRK5-1 was Gram-stained and observed using an optical microscope. The results are as follows: Figure 5 As shown, the strain turns red after Gram staining, indicating that it is a Gram-negative bacterium.
[0079] (3) SEM (Scanning Electron Microscopy) observation
[0080] Selected colonies with good growth from the plate were inoculated onto seed culture medium and incubated overnight for 12 hours at 37℃ and 200 rpm in a shaker. The next day, the bacterial suspension was poured into centrifuge tubes and centrifuged at 4000 rpm for 5 minutes. The supernatant was discarded, and the bacterial precipitate was retained. The wet bacterial suspension at the bottom of the centrifuge tube was mixed with sterile distilled water, and a drop of the resuspension was placed in the center of a silicon wafer pretreated with 75% ethanol. The silicon wafer was then dried in a 45℃ oven until no droplets remained on the surface. The sample was then subjected to three gold sputtering processes using an ion sputtering system to complete the sample preparation, ready for electron microscopy. The results are as follows: Figure 6 As shown, the bacterial cells are short, round rods with flat ends, measuring (0.3-0.8) μm × (0.5-1.5) μm, and are relatively small in size.
[0081] Example 4
[0082] NRK5-1 Molecular Biological Identification
[0083] (1) 16S rDNA sequencing:
[0084] Genomic DNA was extracted from the bacterial strain using the Ezup Columnar Bacterial Genomic DNA Extraction Kit from Sangon Biotech Co., Ltd. Specific procedures are detailed in the instruction manual. Using the extracted genomic DNA as a template, PCR amplification was performed according to the system shown in Table 3 and the procedure shown in Table 4. The PCR products of the 16S rDNA were then subjected to agarose gel electrophoresis. The results are as follows: Figure 7 As shown. The target gene band is bright, and marker analysis indicates its sequence size is approximately 1400 bp. PCR amplification was performed using universal primers, and the sequence is as follows:
[0085] 27F: 5'-AGTTTGATCMTGGCTCAG-3' (SEQ ID NO.2), where M represents A / C;
[0086] 1492R:5'-GGTTACCTTGTTACGACTT-3'(SEQ ID NO.3)
[0087] Table 3 PCR System
[0088]
[0089]
[0090] Table 4 PCR Validation Procedure
[0091]
[0092] The PCR-amplified fragment was transferred to Sangon Biotech (Shanghai) Co., Ltd. for subsequent 16S rDNA sequencing. The results showed that the 16S rDNA of NRK5-1 was 1431 bp in length, and the sequence is shown in SEQ ID NO.1.
[0093] (2) Construction of the phylogenetic tree:
[0094] The 16S rDNA sequence determined by Sangon Biotech was compared with the NCBI database, and the results are as follows: Figure 8 As shown in the figure. The strains with the highest correlation to the sample strains were selected for multiple sequence alignment analysis, and a phylogenetic tree was constructed using MEGA software. NRK5-1 and its closely related strains were ranked and a phylogenetic tree was constructed using MEGA software. The results are shown in the figure. Figure 9 As shown, most strains with 100% 16S rDNA sequence similarity to strain NRK5-1 belong to the genus *Escherichia coli*. *Enterobacter kobei* showed the highest similarity to strain NRK5-1. Based on BLAST homology alignment and phylogenetic tree analysis, this strain was named *Enterobacter kobei* 2020T51 for further research on fermentation conditions.
[0095] Example 5
[0096] Under aseptic conditions, 1 mL of activated seed culture was inoculated into 20 mL of pH 7.5 medium (the components and ratios of the initial fermentation medium are shown in Table 1) at an inoculation ratio of 5% (v / v). Fermentation was carried out at 30℃ and 200 r / min for 12 h. The OD of the bacterial culture was measured after fermentation. 600 The remaining bacterial culture was ultrasonically treated to prepare crude enzyme solution. The ultrasonic cell disruptor was set to 30% power, operating for 3 seconds followed by a 5-second pause, for 8 minutes to disrupt the cell culture. The disrupted cell culture was then centrifuged at 4℃ and 4000 rpm for 20 minutes, and the supernatant obtained was the crude enzyme solution. 50 μL of the crude enzyme solution and 50 μL of the mother solution (prepared as a solution containing 1 mM ATP, 1 mM NR, and 1 mM MgSO4·7H2O, using PBS buffer at pH 7.4 as the solvent) were gently mixed in 1.5 mL of EP solution and incubated at 37℃ for 15 minutes. After incubation, the sample was immediately placed in a 95℃ metal bath for 1 minute to terminate the enzyme conversion reaction. The NMN content in the reaction system was detected by HPLC (specific procedures are the same as in Example 2).
[0097] Example 6
[0098] Optimization of fermentation media
[0099] carbon source
[0100] (1) Based on the initial fermentation medium described in Example 5, 4% of the sucrose in the initial fermentation medium was replaced by six carbon sources with a mass concentration of 2% rhamnose, glucose, fructose, sucrose, galactose, and soluble starch, while keeping the other components and proportions unchanged, to investigate the effect of different carbon sources on NRK expression levels. All other aspects were the same as in Example 5.
[0101] The results are as follows Figure 10 As shown. The OD values of the bacterial cultures corresponding to six carbon sources were observed. 600 It can be seen that fructose and maltose produced the best cell growth, while the growth of the remaining four carbon sources was similar and their OD values were similar. 600 All values were above 0.8. This indicates that Enterobacterkobei 2020T51 has a wide range of carbon source selectivity, and all six carbon sources can be utilized by this strain. The NMN production in the transformation system can reflect the activity of NRK enzyme. Figure 10 The results show that glucose is the most favorable carbon source for NRK expression, while fructose, maltose, soluble starch, and sucrose are slightly less effective. Galactose is the least suitable carbon source for the growth and metabolism of this strain, regardless of OD... 600The NMN content remained at its lowest. Considering the research objective of this experiment was to improve the NRK production enzyme activity of the strain, glucose was selected as the optimal carbon source for the culture medium. The NMN yield corresponding to the glucose carbon source fermentation medium was 198.18 μmol / L, with a substrate conversion rate of 19.81%.
[0102] (2) Based on the optimal carbon source culture medium selected in the above experiment, adjustments were made to use the optimal carbon source with a mass concentration of 0%, 1%, 2%, 3%, 4%, and 5%, respectively, while keeping the other components and proportions unchanged, to investigate the effect of different optimal carbon source concentrations on NRK expression levels. All other aspects were the same as in Example 5.
[0103] The results are as follows Figure 11 As shown, adding a final glucose concentration of 1% to the culture medium promotes NRK expression in the bacteria, and the NMN yield is 1 / 5 higher than that in the medium without glucose. Further increasing the glucose concentration in the medium leads to a decrease in NMN yield. Furthermore, compared to the medium without glucose, the bacterial growth is slightly worse after adding glucose, and the OD of the bacterial solution... 600 The value was approximately 0.9. Considering that the research objective of this experiment was to improve the enzyme activity of NRK production by the strain, a glucose concentration of 1% was chosen as the optimal concentration. At this concentration, the NMN yield in the transformation system was 214.55 μmol / L, and the substrate conversion rate was 21.45%.
[0104] nitrogen source
[0105] (1) Based on the carbon source-optimized fermentation medium, adjustments were made by replacing the nitrogen source in the original medium with 2% (w / w) beef meal, yeast extract, beef extract, multivalent peptone, and tryptone, respectively, while keeping the other components and proportions unchanged, to investigate the effect of different nitrogen sources on NRK expression levels. All other aspects were the same as in Example 5.
[0106] The results are as follows Figure 12As shown, different nitrogen sources had varying effects on the expression level and growth of NRK in wild-type bacteria. Under conditions where the nitrogen source concentration was 2%, urea and ammonium chloride, two inorganic nitrogen sources, were unfavorable for the growth of the strain, and no NMN was detected in the transformation system, indicating that the strain could not utilize inorganic nitrogen sources to produce enzymes. Among organic nitrogen sources, tryptone medium yielded the lowest NMN, making it unsuitable for NMN fermentation. The cell growth and NMN product concentration in beef meal and yeast extract media were at moderate levels. Multivalent peptone and beef extract media showed good cell growth and relatively high NMN yields. The beef extract-containing medium showed the highest NRK expression level. This may be because the drying process of powdered media resulted in the loss of some active ingredients, while the paste-like medium retained more vitamins and other nutrients, allowing the strain to absorb more nutrients to synthesize the target protein. However, considering the viscous paste-like texture of beef extract, it is not convenient to weigh it on a precision balance. Powdered multivalent peptone is easier to quantify during weighing, and as a mixed peptone, it can comprehensively provide the nutrients required for the growth and metabolism of this wild fungus. Therefore, multivalent peptone was ultimately selected as the optimal nitrogen source, with an NMN yield of 206.33 μmol / L and a substrate conversion rate of 20.63% in the corresponding conversion system.
[0107] (2) Based on the optimal nitrogen source culture medium selected in the above experiment, adjustments were made to use the optimal nitrogen source with a mass concentration of 1%, 1.5%, 2%, 2.5%, 3%, and 3.5%, respectively, while keeping the other components and proportions in the culture medium unchanged, in order to investigate the effect of different optimal nitrogen source concentrations on NRK expression levels. All other aspects were the same as in Example 5.
[0108] The results are as follows Figure 13 As shown, different concentrations of nitrogen source had varying effects on the expression level and growth of NRK in wild-type fungi. As the concentration of polyvalent peptone increased from 1% to 3.5%, the OD of the bacterial culture... 600 The nitrogen content first increases and then decreases. The reason for this might be that when the nitrogen source concentration is below 2%, the nitrogen content in the culture medium is insufficient to provide enough nutrients for the bacteria, leading to a decrease in microbial activity; when the nitrogen source concentration is above 2.5%, the reduced carbon-to-nitrogen ratio in the culture medium may shorten the bacterial life cycle and cause premature autolysis, thus resulting in decreased cell OD. 600The NMN production in the conversion system initially increased and then decreased as the concentration of polypeptone increased from 1% to 3.5%. The NMN production peaked at 202.37 μmol / L when the polypeptone concentration was 3%. Further increasing the nitrogen source concentration resulted in a decrease in NMN production. This may be because excessive nitrogen in the culture medium triggers complex metabolic reactions and accumulates unnecessary metabolic waste, affecting intracellular protein synthesis and folding, leading to a decrease in NRK activity. Considering that the research objective of this experiment was to improve the NRK production enzyme activity of the strain, 3% polypeptone was chosen as the optimal nitrogen source concentration in the culture medium, corresponding to an NMN production of 202.37 μmol / L and a substrate conversion rate of 20.23%.
[0109] KH2PO4 content
[0110] In the above experiments, KH2PO4 at concentrations of 0%, 0.25%, 0.5%, 0.75%, 1%, and 1.25% (w / w) was added to the carbon and nitrogen source optimized culture medium to investigate the effect of different concentrations of KH2PO4 on NRK expression levels. All other procedures were the same as in Example 5.
[0111] The results are as follows Figure 14 As shown, as the KH2PO4 concentration increased from 0.25% to 1.5%, bacterial growth and NRK expression levels showed a trend of first increasing and then decreasing. At an added KH2PO4 concentration of 0.75%, the bacterial OD... 600 The concentration reached a peak of 1.523, corresponding to a peak NMN yield of 208.27 μmol / L, with a substrate conversion rate of 20.82%. Therefore, the conclusion of this experiment is that adding 0.75% KH2PO4 to the fermentation medium is most suitable.
[0112] MgSO4·7H2O content
[0113] In the above experiments, MgSO4·7H2O was added to the culture medium optimized for carbon source, nitrogen source, and KH2PO4, with mass concentrations of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, and 0.06%, respectively, to investigate the effects of different concentrations of Mg. 2+ The effect on NRK expression levels. All other aspects are the same as in Example 5.
[0114] The results are as follows Figure 15 As shown, as the concentration of MgSO4·7H2O increased from 0.01% to 0.06%, the bacterial growth and the NRK enzyme activity produced, similar to those of KH2PO4, showed a trend of first increasing and then decreasing. At an added MgSO4·7H2O concentration of 0.03%, the bacterial OD... 600The concentration reached a peak of 1.327 μmol / L, corresponding to a peak NMN yield of 218.245 μmol / L, with a substrate conversion rate of 21.82%. Therefore, the conclusion of this experiment is that adding 0.03% MgSO4·7H2O to the fermentation medium is most suitable.
[0115] All of the above experiments were performed in parallel with two sets of samples for analysis.
[0116] Example 7
[0117] Optimization of fermentation culture conditions
[0118] initial pH
[0119] The pH of the optimized fermentation medium was adjusted to 6.0, 6.5, 7.0, 7.5, and 8.0, respectively, to investigate the effect of different initial pH on NRK expression levels. All other procedures were the same as in Example 5.
[0120] The results are as follows Figure 16 As shown, this wild-type bacterium grows well at pH 6-7. The cell density reaches its maximum at a culture medium pH of 6.5, indicating that the optimal pH for this bacterium is 6.5, and it is more suited to a slightly acidic to neutral environment. Combined with the optimized glucose concentration results, it is speculated that a slightly alkaline environment is unfavorable for the cell's absorption and utilization of carbon sources. It is worth noting that the optimal pH for growth is not necessarily the optimal pH for NRK expression. (Observation) Figure 16 It was observed that as the pH increased from 6 to 8, the NMN production first increased and then decreased. The NMN production peaked at pH 7, possibly because NRK enzyme activity is inhibited at pH levels above 7. Considering the research objective of improving the NRK production activity of the strain, pH 7.0 was selected as the optimal pH for the fermentation medium. At this pH, the NMN production in the conversion system was 282.56 μmol / L, and the substrate conversion rate was 28.25%.
[0121] Shaking speed
[0122] The optimized fermentation medium was adjusted to pH 7.0. Under sterile conditions, 1 mL of activated seed culture was inoculated into 20 mL of medium at an inoculation ratio of 5% (v / v). The culture was incubated at 30℃ on a shaker at speeds of 150 rpm, 180 rpm, 200 rpm, and 220 rpm for 12 h. Samples were then taken to determine the OD of the fermentation broth. 600 The effect of different shaking speeds on NRK expression levels was investigated. The remaining bacterial culture was ultrasonically treated to prepare a crude enzyme solution for enzymatic conversion. The NMN content in the reaction system was detected by HPLC. The specific procedures were the same as in Example 5.
[0123] The results are as follows Figure 17As shown, as the rotation speed increased from 150 r / min to 220 r / min, the biomass of the wild fungus showed an increasing trend, and the OD of the bacterial solution... 600 The increase from 1.0114 to 1.2019 indicates that the bacterium can adapt to higher shear forces. However, with increasing rotation speed, the NMN yield showed a trend of first increasing and then slightly decreasing, reaching a peak of 271.2 μmol / L at a rotation speed of 200 r / min. Increasing the rotation speed to 220 r / min resulted in a slight decrease in NMN yield. This suggests that excessively high aeration is not suitable for the bacterium to produce high levels of NRK expression. It is speculated that this is because high rotation speed improves aeration in the shake flask, leading to vigorous bacterial growth and reproduction, but also the accumulation of certain primary and secondary metabolites, which inhibit the synthesis of the target protein. Therefore, the optimal rotation speed for culturing the strain of this invention can be considered to be 200 r / min. At this speed, the NMN yield in the transformation system reached 271.2 μmol / L, with a substrate conversion rate of 27.12%.
[0124] Fermentation time and temperature
[0125] The optimized fermentation medium was adjusted to the optimal pH 7.0. Under sterile conditions, 1 mL of activated seed culture was inoculated into 20 mL of medium at an inoculation ratio of 5% (v / v). The culture was then incubated for 8 to 24 hours at constant temperature shaking at 25℃, 30℃, 37℃, and 40℃, with samples taken every 4 hours to measure the OD value of the fermentation broth. 600 To investigate the effects of different fermentation times and temperatures on NRK expression levels, the OD values of the bacterial culture were recorded. 600 The remaining bacterial culture was ultrasonically treated to prepare a crude enzyme solution for enzymatic conversion. The NMN content in the reaction system was detected by HPLC. The specific operation was the same as in Example 5.
[0126] The results are as follows Figure 18 As shown, the NMN production in the conversion system at four different temperatures (25, 30, 37, and 40℃) initially increased and then decreased. With the culture temperature increasing from 25℃ to 40℃, the enzyme activity of NRK produced by the bacteria generally showed an increasing trend. At 40℃, the NMN production reached a peak of 332.23 μmol / L after 16 hours of culture, indicating that slightly higher temperatures are more suitable for nicotinamide ribokinase expression. Regarding the growth of the strain, the OD values of the bacterial culture at 25, 30, and 37℃ showed... 600 All increased with increasing fermentation time. However, at 40℃, the bacterial cell density OD of the culture solution increased after 16 hours of fermentation. 600The cell density reached a peak of 1.3, but continued fermentation with longer duration led to a decrease in cell density. This was likely due to the strain aging and undergoing autolysis at the high temperature of 40℃ for an extended period. Considering the research objective of improving the NRK production enzyme activity of the strain, a culture temperature of 40℃ and a fermentation time of 16 hours were selected as the optimal fermentation temperature and time. Under these conditions, the corresponding NMN yield in the conversion system was 332.23 μmol / L, with a substrate conversion rate of 33.22%.
[0127] All of the above experiments were performed in parallel with two sets of samples for analysis.
[0128] As demonstrated in the above embodiments, this invention uses sludge collected from the sewage outlet of the Shanghai Shuze Biotechnology Research Institute to screen NRK-producing bacteria. A strain screening model was established, and the finally screened strain NRK5-1 can convert to produce 142.5 μmol / L NMN, with a substrate conversion rate of 14.25%. Morphological analysis revealed that the colonies were round, milky white, smooth, transparent, with regular edges, and upward convex, with a diameter of less than 2 mm. The bacterial cells were short, rod-shaped, measuring (0.3-0.8) μm × (0.5-1.5) μm, and showed a Gram-negative red stain. Combined with molecular biological identification, strains with 100% homology to strain NRK5-1 were mostly from the genus *Escherichia coli*. The phylogenetic tree construction showed that *Enterobacter kobei* NR113321.1 had the highest similarity to strain NRK5-1. Therefore, strain NRK5-1 was identified as *Enterobacter kobei* and named *Enterobacter kobei2020T51*. The fermentation medium and conditions for strain Enterobacter kobei 2020T51 were optimized. The optimized conditions were: inoculation with a medium containing 1% glucose, 3% polyvalent peptone, 0.75% KH2PO4, and 0.03% MgSO4·7H2O, with an initial pH of 7.0, and incubation at 40℃ and 200 r / min for 16 h. Under these conditions, the strain could convert 332.23 μmol / L NMN, with a substrate conversion rate of 33.22%, which was 2.3 times that before optimization.
[0129] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A type of Kobe Enterobacter ( Enterobacter kobei ), characterized in that, The Kobe Enterobacter was deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 24913.
2. A method for producing NRK, characterized in that, The process includes the following steps: fermenting and culturing the Kobe Enterobacter as described in claim 1, and crushing the obtained fermentation broth to obtain a crude enzyme solution containing NRK.
3. The method according to claim 2, characterized in that, The fermentation medium for the fermentation culture includes 0%-5% carbon source, 1%-3.5% nitrogen source, 0%-1.25% KH2PO4 and 0.01%-0.06% MgSO4·7H2O.
4. The method according to claim 3, characterized in that, The carbon sources include rhamnose, glucose, fructose, sucrose, galactose, and soluble starch.
5. The method according to claim 3, characterized in that, The nitrogen sources include beef meal, yeast extract, beef extract, multivalent peptone, and trypsin.
6. The method according to claim 3, characterized in that, The pH of the culture medium during fermentation is 6.0-8.
0.
7. The method according to claim 2, characterized in that, The fermentation conditions are: 25℃-40℃, shaking culture for 8h-24h.
8. The method according to claim 7, characterized in that, The oscillation culture is a shaking culture on a shaker, and the rotation speed of the shaker is 150 r / min-220 r / min.
9. The use of the Kobe Enterobacter as described in claim 1 in the preparation of NMN.
10. The application according to claim 9, characterized in that, The process includes the following steps: fermenting and culturing the Kobe Enterobacter as described in claim 1, crushing the bacterial solution to obtain a crude enzyme solution, and mixing the crude enzyme solution with a mother liquor containing ATP and NR to obtain NMN.