A denitrogenation bombyx mori microorganism and its application in preparation of high-titer pyrroloquinoline quinone

Abstract

The application discloses a denitrifying hyphomicrobium and application thereof in preparation of high-titer pyrroloquinoline quinone. The denitrifying hyphomicrobium is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20242618, and the preservation date is November 21, 2024. The application further optimizes the adding amount and adding mode of trace elements in the fermentation medium of the denitrifying hyphomicrobium, so that the titer of PQQ can be effectively improved by adjusting the related metabolic enzyme activity in the bacterial growth process and the synthesis and release of the product. The denitrifying hyphomicrobium HXRD10-32 in the application combined with the corresponding fermentation method can effectively improve the production efficiency of PQQ in microbial fermentation, and is beneficial to the efficient development of the PQQ industry.

Description

[0001] Related patents This application is a divisional application of Chinese invention patent application No. 2024119513559, filed on December 26, 2024, entitled "A denitrifying microbacterium producing high-valence pyrroloquinoline quinone and its application". Technical Field

[0002] This application relates to the field of microbial technology, and in particular to a denitrifying microbacterium and its application in the preparation of high-efficiency pyrroloquinoline quinone. Background Technology

[0003] Pyrroloquinoline quinone (PQQ) is a cofactor for oxidoreductases such as methanol dehydrogenase, which participate in electron transport in the respiratory chain, and possesses a variety of unique physiological functions. PQQ's strong antioxidant capacity helps scavenge free radicals in the body, which is beneficial in preventing cardiovascular and cerebrovascular diseases caused by free radicals. In addition, PQQ also has neuroprotective functions, improves mitochondrial performance, and alleviates chronic inflammation. Due to its various beneficial physiological effects on the human body, PQQ has been approved as a safe dietary supplement by the U.S. Food and Drug Administration and the European Food Safety Authority, and has great potential for application development.

[0004] Mammals cannot synthesize PQQ themselves and must obtain it from food sources. The main bacteria capable of synthesizing PQQ are Gram-negative bacteria such as *Methylobacteria*, *Microbes*, and *Pseudomonas*. Early PQQ production primarily relied on chemical synthesis, but this was limited by its complex and cumbersome synthesis steps, numerous byproducts, and high production costs, hindering industrialization. With the application of microbial fermentation technology, the method of preparing PQQ through fermentation of PQQ-producing bacteria offers advantages such as high yield, environmental friendliness, safety, and low cost. The simple culture medium composition also facilitates subsequent separation and purification. Therefore, compared to traditional chemical synthesis methods, it has significant advantages and a greater prospect for the industrialization of PQQ.

[0005] Denitrifying silk microbes ( Hyphomicrobium denitrificans PQQ is a methyltrophic bacterium capable of efficiently synthesizing PQQ, utilizing methanol as its sole carbon and energy source for growth and metabolism. Since the metabolic pathways of microbial PQQ synthesis are not yet fully elucidated, targeted genetic modification of strains to promote PQQ biosynthesis remains a significant challenge. Therefore, methods to increase PQQ yield during fermentation mainly include two aspects: the selection of high-yielding strains and the optimization and control of fermentation conditions.

[0006] To improve H. denitrificansThe production yield of PQQ synthesized by bio-fermentation, through screening and domesticating high-yield PQQ starting strains and optimizing fermentation processes to control production costs and achieve large-scale promotion, has become an urgent problem to be solved. Summary of the Invention

[0007] To solve at least one of the above-mentioned technical problems, the technical solution adopted in this application is as follows.

[0008] The first aspect of this application provides a denitrifying silk microbacterium HXRD10-32 ( Hyphomicrobium denitrificans HXRD10-32), deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20242618, on November 21, 2024.

[0009] This application involves washing and centrifuging fresh sludge containing methanol to collect the supernatant, adding methanol as the sole carbon source for enrichment culture, and selecting suitable strains by detecting the PQQ content in the fermentation broth using high performance liquid chromatography. After multiple rounds of treatment with continuous ultraviolet irradiation and nitrosoguanidine, the denitrifying microbacterium HXRD10-32 was obtained through screening with a screening medium.

[0010] In some specific embodiments of this application, the 16S rDNA sequence of the denitrifying filamentous microorganism HXRD10-32 is shown in SEQ ID No. 1. It can be understood that any 16S rDNA sequence identical to that of the denitrifying filamentous microorganism HXRD10-32 is included within the scope of protection of this application. Preferably, similarities exceeding 99.86% are considered identical, or similarities exceeding 99.9% are considered identical.

[0011] The second aspect of this application provides a microbial agent, including the denitrifying silk-producing microbacterium HXRD10-32 described in the first aspect of this application.

[0012] In some embodiments of this application, the microbial agent is a bacterial solution or powder (e.g., freeze-dried powder) prepared using the denitrifying silk microbacterium HXRD10-32.

[0013] A third aspect of this application provides a fermentation broth comprising the denitrifying filamentous microorganism HXRD10-32 described in the first aspect of this application.

[0014] In this application, the fermentation broth is obtained by fermenting the denitrifying silk microorganism HXRD10-32.

[0015] The fourth aspect of this application provides the application of the denitrifying filamentous microorganism HXRD10-32 described in the first aspect of this application, the microbial agent described in the second aspect of this application, or the fermentation broth described in the third aspect of this application: for the preparation of pyrroloquinoline quinone; or for the preparation of one or more of human drugs, veterinary drugs, health products, food, beverages, cosmetics, additives, and feed containing pyrroloquinoline quinone.

[0016] The fifth aspect of this application provides a method for producing pyrroloquinoline quinone, comprising the following steps: The denitrifying silk-producing microorganism HXRD10-32 described in the first aspect of this application or the microbial agent described in the second aspect of this application is fermented to obtain the pyrroloquinoline quinone.

[0017] In some embodiments of this application, the denitrifying silk microbacterium HXRD10-32 or the bacterial agent is fermented in a fermentation medium containing an assimilated carbon source.

[0018] In some specific embodiments of this application, the assimilable carbon source is methanol or methylamine, or may include other carbon sources besides methanol, wherein the other carbon sources are selected from at least one or any combination of methylamine, ethanol, glycerol, formic acid, acetic acid, mannitol, starch, maltodextrin, glucose, sucrose, lactose, maltose, industrial molasses, soybean oil, and sorbitol.

[0019] In some other specific embodiments of this application, methanol is the sole carbon source.

[0020] In some preferred embodiments of this application, the assimilable carbon source is methanol. Before inoculating the denitrifying filamentous microorganism HXRD10-32 or the bacterial agent, 50% aseptically filtered methanol is added to bring the initial methanol content in the fermentation medium to 4-6 g / L. In some specific embodiments of this application, during fermentation, the carbon source is supplemented by adding 50% aseptically filtered methanol to control the methanol concentration in the fermentation broth to 3-5 g / L.

[0021] In some embodiments of this application, the fermentation medium further includes inorganic salts, which include one or more of Na3C6H5O7, KH2PO4, K2HPO4, Na2HPO4, (NH4)2SO4, CaCO3, FeSO4, ZnSO4, CuSO4, MgSO4, MnSO4, NaCl, KCl, CaCl2, and FeCl3.

[0022] In some preferred embodiments of this application, the fermentation medium is composed of: CH3OH 5 g / L, (NH4)2SO4 2 g / L, KH2PO4 1.5 g / L, Na2HPO4·12H2O 9 g / L, and MgSO4·7H2O 2.0 g / L.

[0023] In some embodiments of this application, a trace element solution is added to the fermentation medium before fermentation and at least once during fermentation. The solutes in the trace element solution include ZnSO4, MnSO4, CuSO4, FeSO4, NaCl, CoCl, CaCl2, and (NH4)6Mo7O. 24 One or more of H3BO3 and KI.

[0024] In some specific embodiments of this application, the trace element solution includes trace element solution A, trace element solution B, trace element solution C, and trace element solution D. Trace element solution A has the following composition: ZnSO4·7H2O 15.0 g / L, MnSO4·H2O 6.5 g / L, CuSO4·5H2O 0.75 g / L, FeSO4·7H2O 12.0 g / L; trace element solution B has the following composition: NaCl 1.5 g / L, CoCl·6H2O 0.05 g / L, CaCl2 22.65 g / L; and trace element solution C has the following composition: (NH4)6Mo7O 24 • 4H₂O 0.05 g / L, H₃BO₃ 0.05 g / L; the trace element D solution consists of KI 0.05 g / L. In some specific embodiments of this application, the concentrations of trace element A, B, C, and D solutions added to the fermentation medium at the initial stage of fermentation are 0.5~2.0 mL / L, respectively. In some specific embodiments of this application, the concentrations of trace element A, B, C, and D solutions added during the acidification process are 0.5~1.0 mL / L, respectively.

[0025] It should be noted that the present invention can adjust the ratio of trace elements, for example, by combining two or more of them, or by splitting one of them into two or more, as long as the content of trace elements in the fermentation medium is comparable to the above scheme.

[0026] In some preferred embodiments of this application, at OD 600 Add trace element solution once when the concentration is >60.

[0027] In some embodiments of this application, dissolved oxygen is controlled to be no less than 30% by adjusting the fermentation speed during the fermentation process.

[0028] In some specific embodiments of this application, the denitrifying silk microbacterium HXRD10-32 or the inoculum agent further includes a step of activation using a seed culture medium before inoculation into the fermentation medium.

[0029] In some specific embodiments of this application, the seed culture medium is composed of CH3OH 10 g / L, (NH4)2SO4 2 g / L, KH2PO4 1.5 g / L, Na2HPO4·12H2O 9 g / L, and MgSO4·7H2O 2 g / L.

[0030] In some specific embodiments of this application, the trace element A, B, C, and D solutions are added to the seed culture medium at a concentration of 0.5 to 1.0 mL / L.

[0031] In some specific embodiments of this application, the activation conditions are 30±1℃ and 150~250r / min, until the fermentation broth OD... 600 =3.0~4.0, activation complete.

[0032] Compared with the prior art, the invention title of this application has the following beneficial effects: This application obtained a denitrifying filamentous microbacterium, HXRD10-32, producing a high-potency pyrroloquinoline quinone through screening and mutagenesis. Hyphomicrobium denitrificans HXRD10-32), deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242618, on November 21, 2024.

[0033] This application further optimizes the amount of trace elements added to the fermentation medium of denitrifying filamentous microorganisms, thereby effectively improving the potency of PQQ by regulating the activity of related metabolic enzymes and the synthesis and release of products during bacterial growth.

[0034] By utilizing the denitrifying raw silk microorganism HXRD10-32 of this application in combination with the corresponding fermentation method, the production efficiency of PQQ by microbial fermentation can be effectively improved, which is conducive to the efficient development of the PQQ industry.

[0035] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0036] The above and other objects, features, and advantages of exemplary embodiments of this application will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of this application are illustrated in the drawings by way of example and not limitation, in which: Figure 1 The phylogenetic tree of the denitrifying silk microbacterium HXRD10-32 in this application is shown.

[0037] Figure 2The HPLC chromatogram of pyrroloquinoline quinone in the supernatant of the denitrified filamentous microorganism fermentation broth in Example 4 of this application is shown.

[0038] Figure 3 The OD detected during the fermentation cycle in Example 4 of this application is shown. 600 and changes in PQQ valence.

[0039] Figure 4 The OD detected during the fermentation cycle in Comparative Example 1 of this application is shown. 600 and changes in PQQ valence.

[0040] Figure 5 The OD detected during the fermentation cycle in Comparative Example 2 of this application is shown. 600 and changes in PQQ valence.

[0041] Figure 6 The OD detected during the fermentation cycle in Comparative Example 3 of this application is shown. 600 and changes in PQQ valence.

[0042] Figure 7 The OD detected during the fermentation cycle in Comparative Example 4 of this application is shown. 600 and changes in PQQ valence.

[0043] Figure 8 The OD detected during the fermentation cycle in Comparative Example 5 of this application is shown. 600 and changes in PQQ valence.

[0044] Figure 9 The OD detected during the fermentation cycle in Comparative Example 6 of this application is shown. 600 and changes in PQQ valence. Detailed Implementation

[0045] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, in particular the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0046] The numerical ranges used in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. The numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided there is an interval of at least two units between any lower and any higher value. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly described in this application.

[0047] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0048] To make the technical problems, technical solutions and beneficial effects solved by this application clearer, the following detailed description is provided in conjunction with embodiments.

[0049] The following examples are used to illustrate preferred embodiments of this application. Those skilled in the art will understand that the techniques disclosed in the examples represent technologies discovered by the inventors that can be used to implement this application, and therefore can be considered preferred embodiments of this application. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of this application.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and all materials cited herein and referenced by them are incorporated herein by reference.

[0051] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0053] Example 1: Screening of PQQ-producing bacteria This embodiment screens PQQ-producing bacteria from a chemical water tank. The specific steps are as follows: (1) Take 5 g of fresh sludge from the chemical wastewater pond, shake it thoroughly in 10 mL of sterile water, let it stand, take 1 mL of the upper suspension and inoculate it into enrichment medium 1 at a 2% inoculation rate. Cultivate at 30℃ and 220 rpm for 5 days to obtain enriched fermentation broth 1. Increase the methanol content in the medium by the same method. Transfer enriched fermentation broth 1 to enrichment medium 2 and enrichment medium 3 in sequence and enrich them at 30℃ and 220 rpm for 5 days. After each transfer, select the fermentation broth that has deepened in color and turned red, centrifuge it and take the supernatant. Detect the PQQ titer in the fermentation broth by high performance liquid chromatography.

[0054] (2) The enriched fermentation broth with the highest PQQ titer was selected, serially diluted, and spread on enrichment plates. It was cultured at 30℃ for 5 days. Single colonies with good growth and clear edges were selected and transferred to screening medium. The culture was carried out at 30℃ and 220 rpm for 6 days. During the culture, the methanol content was measured on the second and fourth days. Methanol was added when the methanol content was low. After the culture was completed, the PQQ titer in the fermentation broth was determined by high performance liquid chromatography. The strain with the highest titer was selected and recorded as HX-SX-81, which was used for mutagenesis treatment.

[0055] The culture medium components described in this embodiment include: 1. Basal culture medium: (NH4)2SO4 2.0 g / L, KH2PO4 1.5 g / L, Na2HPO4·12H2O 9.0 g / L, MgSO4·7H2O 2.0 g / L, CaCO3 1.0 g / L, trace element solution 0.7 mL / L; 2. Enrichment medium 1 Add 4 g / L methanol as a carbon source to the basic culture medium.

[0056] 3. Enrichment medium 2 Add 6 g / L methanol as a carbon source to the basic culture medium.

[0057] 4. Enrichment medium 3 Add 8 g / L methanol as a carbon source to the basic culture medium.

[0058] 5. Enrichment Plate Add 20 g / L agar powder and 10 g / L methanol as a carbon source to the basic culture medium.

[0059] 6. Screening culture medium 20 g / L methanol was added as a carbon source to the basal culture medium; the micronutrient solution consisted of ZnSO4·7H2O 15.0 g / L, MnSO4·H2O 6.5 g / L, CuSO4·5H2O 0.75 g / L, FeSO4·7H2O 12.0 g / L, NaCl 1.5 g / L, CoCl·6H2O 0.05 g / L, CaCl2 22.65 g / L, and (NH4)6Mo7O 24 ·4H2O 0.05 g / L, H3BO30.05 g / L, KI 0.05 g / L.

[0060] Example 2: Mutagenesis and Breeding of High-Productivity PQQ Bacteria This embodiment provides a method for breeding high-producing PQQ bacteria through a combination of physicochemical mutagenesis using high-concentration methanol as an antagonist. The specific steps are as follows: (1) Using strain HX-SX-81 obtained in Example 1 as the original strain, it was inoculated into the screening medium and cultured until the seed culture OD 600 Value 1.6. Take 5 mL of bacterial suspension, centrifuge to remove the supernatant, wash twice with PBS buffer, and resuspend. Take 2 mL of bacterial suspension and place it under a sterile plate. Place the plate at 30 cm from a preheated 25W UV lamp and irradiate with a sterile magnetic bead at 120 rpm for 20–60 s (e.g., 20 s, 30 s, 40 s, 50 s, 60 s). Add nitrosoguanidine to the UV-irradiated bacterial suspension at a concentration of 1–5 mg / L (e.g., 1 mg / L, 2 mg / L, 3 mg / L), mix well, and incubate at 30°C in the dark for 60 min. Centrifuge for 10 min, remove the supernatant, wash twice with PBS, and resuspend. Dilute the UV-Nitroguanidine combined mutagenesis-treated bacterial suspension and the untreated bacterial suspension appropriately, spread them on screening plates, and incubate at 30°C in the dark for 8 days.

[0061] (2) After the culture was completed, the lethality was calculated based on the number of colonies on the untreated plates and plates with different mutagenic doses. Single colonies with good growth and clear colony edges were selected from plates with a lethality of 90% and inoculated into the screening medium. The culture was carried out at 30 ℃ and 220 rpm for 6 days. During the culture, methanol was added every two days according to the remaining methanol in the medium. After the culture was completed, the PQQ titer in the fermentation broth was determined by high performance liquid chromatography. Strains with higher PQQ titers were selected for a new round of compound mutagenesis, and the process was repeated three times.

[0062] (3) After the three rounds of combined mutagenesis, the bacterial suspension was spread on screening plates and cultured at 30 °C for 8 days. Single colonies with good growth and clear edges were selected and inoculated into seed shake flask culture medium and cultured at 30 °C and 220 rpm for 62 h until the OD of the seed culture was reached. 600 The PQQ titer was 3.5. 1.5% of the culture medium was inoculated into a fermentation shake flask at 30℃ and 220 rpm for 6 days. During the culture, methanol was added every 2 days until the methanol concentration reached 5 g / L. After the culture was completed, the PQQ titer in the fermentation broth was detected by high performance liquid chromatography.

[0063] Using the above steps, a high-yielding PQQ strain was obtained, numbered HX-YB-32, which reached a titer of 127.2 mg / L after 6 days of shake-flask fermentation.

[0064] The screening plates and screening media described in this embodiment are the same as the enrichment plates and screening media described in Example 1. The fermentation shake flask medium is the basic medium described in Example 1 with 5 g / L methanol added as the sole carbon source.

[0065] Example 3: Identification and Preservation of Strains The mutant strain HX-YB-32 obtained in Example 2 was subjected to physiological and biochemical index detection, morphological observation and species identification.

[0066] The results showed that the strain was an aerobic bacterium, negative for methyl red, VP, and starch hydrolysis tests, positive for oxidase, capable of reducing nitrite, and did not produce indole or hydrogen sulfide. It could utilize methanol, arabinose, and trehalose, was sensitive to tetracycline, but not to ampicillin, kanamycin, or chloramphenicol. The optimal growth temperature was 28–32℃, pH 6–8, the colonies were small and milky white with a smooth surface, and Gram staining was negative.

[0067] Using DNA from the mutant strain HX-YB-32 as a template, PCR amplification was performed using universal 16S rDNA primers. The amplified product was sent to Qingdao NIO Biotechnology for sequencing, and the 16S rDNA sequence of this strain was obtained as follows (SEQ ID No. 1): The 16S rDNA sequence of this strain was compared with the online BLAST sequence in the NCBI database. H. denitrificans strain ATCC 51888 H. denitrificans The strain DSM 1869 showed a phylogenetic relationship of over 99%. Phylogenetic tree construction also revealed that this strain was related to... H. denitrificans Divided into the same branch (e.g.) Figure 1 (as shown), therefore the strain was identified and named H. denitrificans HXRD10-32.

[0068] This strain was deposited on November 21, 2024, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province; accession number: CCTCC NO: M 20242618.

[0069] Example 4: Production of PQQ by denitrifying raw silk microorganisms through fermentation This embodiment utilizes H. denitrificans HXRD10-32 is used for fermentation to produce PQQ, including the following steps: (1) Culture medium ① Seed culture medium The seed culture medium consisted of (NH4)2SO4 2 g / L, KH2PO4 1.5 g / L, Na2HPO4·12H2O 9 g / L, MgSO4·7H2O 2 g / L, trace element solution A 0.7 mL / L, trace element solution B 0.7 mL / L, trace element solution C 0.7 mL / L, and trace element solution D 0.7 mL / L. The solution was brought to a final volume of 100 mL and transferred to a 500 mL Erlenmeyer flask. After sterilization at 121 °C for 20 min, methanol was added to a final concentration of 10 g / L.

[0070] ② Fermentation medium The fermentation medium consisted of (NH4)2SO4 2 g / L, KH2PO4 1.5 g / L, Na2HPO4·12H2O 9 g / L, MgSO4·7H2O 2.0 g / L, trace element solution A 0.7 mL / L, trace element solution B 0.7 mL / L, trace element solution C 0.7 mL / L, and trace element solution D 0.7 mL / L. The mixture was brought to a final volume of 9 L and transferred to a 15 L fermenter. After sterilization at 121℃ for 20 min, methanol was added to a final concentration of 5 g / L.

[0071] In the above seed culture medium and fermentation culture medium, the composition of trace element solution A is ZnSO4·7H2O 15.0 g / L, MnSO4·H2O 6.5 g / L, CuSO4·5H2O 0.75 g / L, FeSO4·7H2O 12.0 g / L; the composition of trace element solution B is NaCl 1.5 g / L, CoCl·6H2O 0.05 g / L, CaCl2 22.65 g / L; and the composition of trace element solution C is (NH4)6Mo7O 24 • 4H2O 0.05 g / L, H3BO3 0.05 g / L; the composition of trace element D solution is KI 0.05 g / L.

[0072] (2) Seed culture Pick H. denitrificans HXRD10-328 glycerol bacteria were thawed at room temperature. The glycerol bacterial suspension was inoculated into seed culture medium at 0.6%. After inoculation, the culture was carried out on a shaker at 30 ℃ and 200 rpm until OD reached. 600 Value 3.5 is available.

[0073] (3) Fermentation in a 15L fermenter After the seeds have been cultured and confirmed to be free of contaminants under a microscope, they are inoculated into a 15 L fermenter containing fermentation medium at an inoculation rate of 10% for fermentation.

[0074] (4) Fermentation process control in 15 L tanks During the fermentation cycle, the fermentation tank volume was 9 L, the culture temperature was 30℃, the initial stirring speed was 150 rpm, the air flow rate was 1.0 VVM, the pH value of the ammonia water was automatically controlled at 6.8, and the culture cycle was 240 h.

[0075] (5) Dissolved oxygen control in 15 L fermentation tank Dissolved oxygen levels are maintained above 30% throughout the fermentation cycle using a stirring mechanism.

[0076] (6) Methanol control in 15 L fermentation tank Fermentation was controlled by feeding methanol. At the beginning of fermentation, 50% methanol was added to the fermentation medium to a methanol concentration of 5.0 g / L before inoculation. As the cells grew, methanol was continuously consumed. The methanol concentration in the fermentation broth was monitored at regular intervals during the fermentation cycle. The methanol concentration in the fermentation broth was controlled to be 3-5 g / L by adjusting the flow rate of 50% methanol.

[0077] (7) Trace element control in 15 L tank fermentation Fermentation was controlled by supplementing with trace element solutions. At the start of fermentation, the initial concentrations of trace elements A, B, C, and D in the culture medium were 1.0 mL / L. The OD of the fermentation broth was measured as the cells grew. 600It begins to grow, during the fermentation process, OD 600 When the concentration is >60, add trace element solution once. The amount of trace element A, B, C and D solution added is 0.5 mL / L.

[0078] (8) Detection of fermentation effect in 15 L tank Throughout the fermentation cycle, methanol content and OD are monitored. 600 The PQQ potency was tested, and the methanol content was determined using an ethanol flow meter. OD 600 The potency of PQQ was determined by spectrophotometry and by high-performance liquid chromatography (HPLC). The HPLC conditions were as follows: TCI Kaseisorb LC ODS 2000 column, injection volume 20 μL, column temperature 30 ℃, flow rate 1.0 mL / min, mobile phase A was a mixture of 10 mmol / L dipotassium hydrogen phosphate and 15 mmol / L tetrabutylammonium bromide (pH 7.4), and mobile phase B was acetonitrile. Gradient elution was used with the following program: 75%A + 25%B (0 min), 75%A + 25%B (25 min), 79%A + 21%B (48 min), 75%A + 25%B (50 min), 75%A + 25%B (25 min), detection wavelength 250 nm. The elution time of PQQ was approximately 14.55 min (e.g., ...). Figure 2 (As shown). Fermentation cycle OD 600 Changes in PQQ valence, such as Figure 3 As shown.

[0079] In this embodiment, the PQQ titer is 3.25 g / L, and the OD... 600 It is 113.20.

[0080] Comparison Example 1 The difference between this comparative example and Example 4 is that the initial addition amount of trace elements A, B, C, and D in the fermentation medium is 0.5 mL / L, while the rest is the same as in Example 4.

[0081] Throughout the fermentation cycle, methanol content and OD are monitored. 600 The PQQ potency was tested, and during fermentation, 50% methanol after sterile filtration was added as a carbon source. OD 600 The results of the entire PQQ titer test are as follows: Figure 4 As shown, after a fermentation period of 240 h, the PQQ titer was 2.35 g / L, and the OD... 600 It is 73.10.

[0082] Comparative Example 2 The difference between this comparative example and Example 4 is that the initial addition amount of trace elements A, B, C, and D in the fermentation medium is 1.3 mL / L, while the rest is the same as in Example 4.

[0083] Throughout the fermentation cycle, methanol content and OD are monitored. 600 The PQQ potency was tested, and during fermentation, 50% methanol after sterile filtration was added as a carbon source. OD 600 The results of the entire PQQ titer test are as follows: Figure 5 As shown, after a fermentation period of 240 h, the PQQ titer was 2.47 g / L, and the OD... 600 It is 127.60.

[0084] Comparative Example 3 The difference between this comparative example and Example 4 is that the initial addition amount of trace elements A, B, C, and D in the fermentation medium is 1.6 mL / L, while the rest is the same as in Example 4.

[0085] Throughout the fermentation cycle, methanol content and OD are monitored. 600 The PQQ potency was tested, and during fermentation, 50% methanol after sterile filtration was added as a carbon source. OD 600 The results of the entire PQQ titer test are as follows: Figure 6 As shown, after a fermentation period of 240 h, the PQQ titer was 2.13 g / L, and the OD... 600 It is 139.60.

[0086] Comparative Example 4 The difference between this comparative example and Example 4 is that the initial addition amount of trace elements A, B, C, and D in the fermentation medium is 2.0 mL / L, while the rest is the same as in Example 4.

[0087] Throughout the fermentation cycle, methanol content and OD are monitored. 600 The PQQ potency was tested, and during fermentation, 50% methanol after sterile filtration was added as a carbon source. OD 600 The results of the entire PQQ titer test are as follows: Figure 7 As shown, after a fermentation period of 240 h, the PQQ titer was 1.98 g / L, and the OD... 600 It is 126.60.

[0088] Comparative Example 5 The difference between this comparative example and Example 4 is that, except for the initial addition of trace element solution to the fermentation medium, no trace element solution is added during the fermentation cycle; otherwise, it is the same as Example 4.

[0089] Throughout the fermentation cycle, methanol content and OD are monitored. 600 The PQQ potency was tested, and during fermentation, 50% methanol after sterile filtration was added as a carbon source. OD 600 The results of the entire PQQ titer test are as follows: Figure 8 As shown, after a fermentation period of 240 h, the PQQ titer was 2.98 g / L, and the OD...600 It is 109.20.

[0090] Comparative Example 6 The difference between this comparative example and Example 4 is that, in addition to the initial addition of trace element solution to the fermentation medium, trace element solution was added once during the fermentation process when OD600>60. The amount of trace element A, B, C and D solutions added was 1.0 mL / L.

[0091] Throughout the fermentation cycle, methanol content and OD are monitored. 600 The PQQ potency was tested, and during fermentation, 50% methanol after sterile filtration was added as a carbon source. OD 600 The results of the entire PQQ titer test are as follows: Figure 9 As shown, after a fermentation period of 240 h, the PQQ titer was 2.77, and the OD... 600 It is 107.40.

[0092] Compared to Example 4, Comparative Examples 1 to 6 all showed lower final fermentation results, as shown in Table 1.

[0093] Table 1. Effects of different trace element additions on PQQ potency

[0094] By comparing Comparative Example 1 and Example 4, it can be seen that the initial addition amount of trace elements significantly affects bacterial growth. In Comparative Example 1, the initial addition amount was too low, impacting bacterial growth and reproduction. OD 600 Significantly lower than Example 4.

[0095] By comparing Comparative Examples 2 to 4 with Example 4, it can be seen that excessive addition of trace elements is not conducive to product synthesis, especially in Comparative Example 4, where excessive initial addition of trace elements resulted in a severely low potency.

[0096] By comparing Comparative Example 5 and Comparative Example 4 with Example 4, it can be seen that supplementing trace elements once during fermentation is beneficial to product synthesis, but excessive supplementation will also affect product synthesis.

[0097] The above results indicate that optimizing the initial trace element content in the fermentation medium and supplementing it with appropriate amounts of trace elements during the fermentation cycle can effectively promote fermentation. H. denitrificans HXRD10-328 cell growth and metabolism, and can significantly improve OD 600 Value and PQQ valence.

[0098] All references to this application are incorporated herein by reference as if each reference were individually incorporated herein by reference. Furthermore, it should be understood that after reading the foregoing teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A denitrifying silk-producing microbacterium ( Hyphomicrobium denitrificans HXRD10-32, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20242618, on November 21, 2024.

2. A microbial agent, characterized in that, Includes the denitrifying silk-producing microbacterium HXRD10-32 as described in claim 1.

3. A fermentation broth, characterized in that, Includes the denitrifying silk-producing microbacterium HXRD10-32 as described in claim 1.

4. The application of the denitrifying microbial strain HXRD10-32 as described in claim 1, the bacterial agent as described in claim 2, or the fermentation broth as described in claim 3 in the preparation of high-efficiency pyrroloquinoline quinone.

5. The application according to claim 4, characterized in that, The application includes the following steps: fermenting and culturing the denitrifying filamentous microorganism HXRD10-32 or the bacterial agent to extract the pyrrolquinoline quinone, or directly extracting the pyrrolquinoline quinone from the fermentation broth.

6. The application according to claim 5, characterized in that, The fermentation culture steps include: thawing the glycerol bacteria of the denitrified filamentous microorganism HXRD10-32 at room temperature, inoculating the glycerol bacterial solution at 0.6% into the seed culture medium, and then culturing it in a shaker at 30 ℃ and 200 rpm until the OD600 value is 3.

5. After the cultured seeds were confirmed to be free of contaminants under microscopic examination, they were inoculated at a 10% inoculum into a 15 L fermenter containing fermentation medium for fermentation. The seed culture medium consisted of (NH4)2SO4 2 g / L, KH2PO4 1.5 g / L, Na2HPO4·12H2O 9 g / L, MgSO4·7H2O 2 g / L, trace element A solution 0.7 mL / L, trace element B solution 0.7 mL / L, trace element C solution 0.7 mL / L, and trace element D solution 0.7 mL / L. The fermentation medium consisted of (NH4)2SO4 2 g / L, KH2PO4 1.5 g / L, Na2HPO4·12H2O 9 g / L, MgSO4·7H2O 2.0 g / L, trace element A solution 0.7 mL / L, trace element B solution 0.7 mL / L, trace element C solution 0.7 mL / L, and trace element D solution 0.7 mL / L. At the start of fermentation, the initial concentration of trace element solutions A, B, C, and D in the culture medium was 1.0 mL / L. During fermentation, when OD600 > 60, trace element solutions were added once, with each of the trace element solutions A, B, C, and D added at a concentration of 0.5 mL / L. The composition of trace element solution A is: ZnSO4·7H2O 15.0 g / L, MnSO4·H2O 6.5 g / L, CuSO4·5H2O 0.75 g / L, FeSO4·7H2O 12.0 g / L; the composition of trace element solution B is: NaCl 1.5 g / L, CoCl·6H2O 0.05 g / L, CaCl2 22.65 g / L; the composition of trace element solution C is: (NH4)6Mo7O 24 • 4H2O 0.05 g / L, H3BO3 0.05 g / L; the composition of trace element D solution is KI 0.05 g / L.

7. The application as described in claim 6, characterized in that, The fermentation process includes methanol replenishment control.

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