Denitrification hyphomicrobe capable of producing high-titer pyrroloquinoline quinone and application of denitrification hyphomicrobe

Abstract

The invention discloses denitrifying hyphomicrobe capable of producing high-titer pyrroloquinoline quinone and an application of the denitrifying hyphomicrobe. The denitrifying hyphomicrobe is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20242618, and the preservation date is November 21, 2024. According to the application, the titer of the PQQ can be effectively improved by further optimizing the addition amount and the addition mode of the trace elements in the denitrifying hyphomicrobium fermentation culture medium and adjusting the activity of related metabolic enzymes in the growth process of bacteria and synthesis and release of products. The denitrifying hyphomicrobe HXRD10-32 is combined with a corresponding fermentation method, so that the PQQ production efficiency of microbial fermentation can be effectively improved, and the efficient development of the PQQ industry is facilitated.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a denitrifying mycelial microbe capable of producing high-efficiency pyrroloquinoline quinone and its application. Background Art

[0002] Pyrroloquinoline quinone (PQQ) is a cofactor of oxidoreductases such as methanol dehydrogenase that participate in electron transfer in the respiratory chain, and has a variety of unique physiological functions. PQQ's strong antioxidant capacity helps to remove free radicals from the human body and is beneficial for preventing cardiovascular and cerebrovascular diseases caused by free radicals; in addition, PQQ also has physiological functions such as neuroprotection, improving mitochondrial performance, and relieving chronic inflammation. Due to its various beneficial physiological effects on the human body, PQQ has been approved by the U.S. Food and Drug Administration and the European Food Safety Authority as a safe dietary supplement, and has great potential for application development.

[0003] Mammals cannot synthesize PQQ by themselves and need to ingest it from food sources. Gram-negative bacteria such as Methylobacterium, Mycena, and Pseudomonas are the main ones that can synthesize PQQ by themselves. In the early days, the production of PQQ was mainly based on chemical synthesis, but it was limited by factors such as cumbersome and complex synthesis steps, many by-products, and high production costs, which was not conducive to industrialization. With the application of microbial fermentation technology, the preparation of PQQ by fermenting PQQ-producing bacteria has the characteristics of high yield, green and harmless, safety, and low cost. The simple composition of the culture medium is also conducive to subsequent separation and purification. Therefore, compared with traditional chemical synthesis methods, it has greater advantages and has greater development prospects in the industrialization of PQQ.

[0004] Denitrifying hyphae Hyphomicrobium denitrificans ) is a methylotrophic bacterium that can efficiently synthesize PQQ and can use methanol as the sole carbon source and energy source for growth metabolism. Since the metabolic pathway of microbial synthesis of PQQ has not been fully elucidated, there are still great challenges in the targeted genetic modification of bacteria to promote the biosynthesis of PQQ. Therefore, the methods to increase the yield of PQQ during fermentation mainly include the selection of high-yield strains and the optimization and regulation of fermentation conditions.

[0005] To improve H. denitrificans The yield of PQQ synthesized by biological fermentation has become an urgent problem to be solved by screening and domesticating high-yield PQQ starting strains and combining them with the optimization of fermentation processes to control the production costs of enterprises and achieve large-scale promotion. Summary of the invention

[0006] In order to solve at least one of the above technical problems, the technical solution adopted in this application is as follows.

[0007] The first aspect of the present application provides a denitrifying hyphae microbacterium HXRD10-32 ( Hyphomicrobium denitrificans HXRD10-32), deposited in China Center for Type Culture Collection, with the deposit number being CCTCC NO: M20242618, and the deposit date being November 21, 2024.

[0008] The present application involves washing and centrifuging fresh sludge containing methanol to collect the supernatant, adding methanol as the sole carbon source for enrichment culture, detecting the PQQ content in the fermentation broth by high performance liquid chromatography to select a suitable strain, and after multiple rounds of continuous ultraviolet irradiation and nitrosoguanidine treatment, screening with a screening medium to obtain the denitrifying hyphae microbacterium HXRD10-32.

[0009] In some specific embodiments of the present application, the 16S rDNA sequence of the denitrifying hyphae microbacterium HXRD10-32 is shown in SEQ ID No. 1. It can be understood that as long as the 16S rDNA sequence is consistent with the denitrifying hyphae microbacterium HXRD10-32, it is included in the protection scope of the present application. Preferably, the similarity (Identities) is more than 99.86% and is considered to be identical, or the similarity is more than 99.9% and is considered to be identical.

[0010] The second aspect of the present application provides a bacterial agent, including the denitrifying hyphae microbacterium HXRD10-32 described in the first aspect of the present application.

[0011] In some embodiments of the present application, the bacterial agent is a bacterial liquid or bacterial powder (such as freeze-dried powder) prepared using the denitrifying hyphae microbacterium HXRD10-32.

[0012] The third aspect of the present application provides a fermentation broth, comprising the denitrifying hyphae microbacterium HXRD10-32 described in the first aspect of the present application.

[0013] In the present application, the fermentation broth is obtained by fermenting the denitrifying hyphae microbacterium HXRD10-32.

[0014] The fourth aspect of the present application provides the use of the denitrifying hyphae microbacterium HXRD10-32 described in the first aspect of the present application, the bacterial agent described in the second aspect of the present application, or the fermentation broth described in the third aspect of the present application: for preparing pyrroloquinoline quinone; or for preparing one or more of human medicines, veterinary drugs, health products, foods, beverages, cosmetics, additives, and feeds containing pyrroloquinoline quinone.

[0015] A fifth aspect of the present application provides a method for producing pyrroloquinoline quinone, comprising the following steps: The denitrifying hyphae microbacterium HXRD10-32 described in the first aspect of the present application or the bacterial agent described in the second aspect of the present application is fermented and cultured to obtain the pyrroloquinoline quinone.

[0016] In some embodiments of the present application, the denitrifying hyphae microbacterium HXRD10-32 or the bacterial agent is fermented in a fermentation medium containing an assimilable carbon source.

[0017] In some specific embodiments of the present application, the assimilable carbon source is methanol, or methylamine, or other carbon sources in addition to methanol, and the other carbon sources are at least selected from methylamine, ethanol, glycerol, formic acid, acetic acid, mannitol, starch, maltodextrin, glucose, sucrose, lactose, maltose, industrial molasses, soybean oil, sorbitol, or any combination of several of them.

[0018] In other specific embodiments of the present application, methanol is the sole carbon source.

[0019] In some preferred embodiments of the present application, the assimilable carbon source is methanol, and 50% methanol after sterile filtration is added before inoculating the denitrifying hyphae microbacterium HXRD10-32 or the bacterial agent, so that the initial methanol content in the fermentation medium is 4-6 g / L. In some specific embodiments of the present application, the carbon source is supplemented by adding 50% methanol after sterile filtration during the fermentation process, and the methanol concentration in the fermentation broth is controlled to be 3-5 g / L.

[0020] In some embodiments of the present application, the fermentation medium also 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.

[0021] In some preferred embodiments of the present application, the composition of the fermentation medium is: CH3OH 5 g / L, (NH4)2SO4 2 g / L, KH2PO4 1.5 g / L, Na2HPO4·12H2O 9 g / L, MgSO4·7H2O 2.0 g / L.

[0022] In some embodiments of the present application, a trace element solution is added to the fermentation medium before fermentation, and the trace element solution is added at least once during the fermentation process. The solutes in the trace element solution include ZnSO4, MnSO4, CuSO4, FeSO4, NaCl, CoCl, CaCl2, (NH4)6Mo7O 24 , H3BO3 and KI.

[0023] In some specific embodiments of the present application, the trace element liquid includes trace element A liquid, trace element B liquid, trace element C liquid and trace element D liquid, wherein the trace element A liquid is composed 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; the trace element B liquid is composed of NaCl 1.5 g / L, CoCl·6H2O 0.05 g / L, CaCl2 22.65 g / L; the trace element C liquid is composed of (NH4)6Mo7O 24 4H2O 0.05 g / L, H3BO3 0.05 g / L; the trace element D liquid composition is KI 0.05 g / L. In some specific embodiments of the present application, the concentrations of trace elements A, B, C, and D liquid added to the fermentation medium at the initial stage of fermentation are 0.5-2.0 mL / L, respectively. In some specific embodiments of the present application, the concentrations of trace elements A, B, C, and D liquid added during the sour process are 0.5-1.0 mL / L, respectively.

[0024] It should be noted that the present invention can adjust the ratio of trace elements, for example, combining two or more of them, or splitting one of them into two or more, as long as the content of trace elements in the protective fermentation medium is equivalent to that in the above scheme.

[0025] In some preferred embodiments of the present application, in OD 600 When the temperature is higher than 60, add trace element solution once.

[0026] In some embodiments of the present application, during the fermentation process, the dissolved oxygen is controlled to be not less than 30% by adjusting the rotation speed.

[0027] In some specific embodiments of the present application, the denitrifying hyphae microbacterium HXRD10-32 or the bacterial agent further comprises a step of activating the microbacterium using a seed culture medium before inoculating the microbacterium into the fermentation medium.

[0028] In some specific embodiments of the present application, the seed culture medium composition is CH3OH 10 g / L, (NH4)2SO4 2 g / L, KH2PO4 1.5 g / L, Na2HPO4·12H2O 9 g / L, MgSO4·7H2O 2 g / L.

[0029] In some specific embodiments of the present application, the trace elements A, B, C, and D solutions are added to the seed culture medium in an amount of 0.5 to 1.0 mL / L.

[0030] In some specific embodiments of the present application, the activation conditions are 30±1°C, 150-250r / min, until the fermentation liquid OD 600 =3.0~4.0, activation is completed.

[0031] Compared with the prior art, the invention of this application has the following beneficial effects: The present invention obtains a denitrifying mycelial microorganism HXRD10-32 (HXRD10-32) that produces high-efficiency pyrroloquinoline quinone by screening and mutagenesis. Hyphomicrobium denitrificans HXRD10-32), deposited in China Center for Type Culture Collection, with the deposit number being CCTCC NO: M 20242618, and the deposit date being November 21, 2024.

[0032] The present application further optimizes the amount of trace elements added to the fermentation medium of denitrifying mycelial 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.

[0033] The use of the denitrifying hyphae microbacterium HXRD10-32 of the present application in combination with a corresponding fermentation method can effectively improve the PQQ production efficiency of microbial fermentation, which is conducive to the efficient development of the PQQ industry.

[0034] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] By reading the detailed description below with reference to the accompanying drawings, the above and other purposes, features and advantages of the exemplary embodiments of the present application will become readily understood. In the accompanying drawings, several embodiments of the present application are shown in an exemplary and non-limiting manner, wherein: Figure 1 The phylogenetic tree of Denitrifying Hyphomicrobium HXRD10-32 in the present application is shown.

[0036] Figure 2 The HPLC spectrum of pyrroloquinoline quinone in the supernatant of the fermentation broth of Denitrifying Mycena Microbacterium in Example 4 of the present application is shown.

[0037] Figure 3 The OD values ​​detected during the fermentation cycle in Example 4 of the present application are shown. 600 And PQQ titer changes.

[0038] Figure 4 The OD detected during the fermentation cycle in Comparative Example 1 of the present application is shown. 600 And PQQ titer changes.

[0039] Figure 5 The OD detected during the fermentation cycle in Comparative Example 2 of the present application is shown. 600 And PQQ titer changes.

[0040] Figure 6 The OD detected during the fermentation cycle in Comparative Example 3 of the present application is shown. 600 And PQQ titer changes.

[0041] Figure 7 The OD detected during the fermentation cycle in Comparative Example 4 of the present application is shown. 600 And PQQ titer changes.

[0042] Figure 8 The OD detected during the fermentation cycle in Comparative Example 5 of the present application is shown. 600 And PQQ titer changes.

[0043] Fig. 9 The OD detected during the fermentation cycle in Comparative Example 6 of the present application is shown. 600 And PQQ titer changes. DETAILED DESCRIPTION

[0044] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the tests and characterization methods used are all synchronized with the filing date of this application. Where applicable, the contents of any patent, patent application or disclosure involved in this application are fully incorporated herein by reference, and their equivalent patent families are also introduced as references, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0045] Numerical ranges in this application are approximate values, so unless otherwise specified, they may include values ​​outside the range. Numerical ranges include all values ​​from the lower limit to the upper limit in increments of 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For a range containing a value less than 1 or containing a fraction greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately considered to be 0.0001, 0.001, 0.01 or 0.1. For a range containing a single digit less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are only specific examples of what is intended to be expressed, and all possible combinations of the values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0046] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, adjuvants or compounds. On the contrary, except for those necessary for operating performance, the term "essentially consisting of..." excludes any other components, steps or processes from the scope of any description of the term below. The term "consisting of..." does not include any components, steps or processes that are not specifically described or listed. Unless explicitly stated, the term "or" refers to the listed individual members or any combination thereof.

[0047] In order to make the technical problems, technical solutions and beneficial effects solved by the present application more clearly understood, the present application is further described in detail below in conjunction with embodiments.

[0048] The following examples are used to demonstrate the preferred embodiments of the present application. It will be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present application, and therefore can be considered as preferred embodiments of the present application. However, it should be understood by those skilled in the art based on this specification that many modifications can be made to the specific embodiments disclosed herein, and still the same or similar results can be obtained without departing from the spirit or scope of the present application.

[0049] 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 belongs, and the disclosures and materials cited therein are hereby incorporated by reference.

[0050] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0051] The experimental methods in the following examples are all 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 are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0052] Example 1 Screening of PQQ-producing bacteria In this example, PQQ-producing bacteria were screened from a chemical water pool. The specific steps are as follows: (1) Take 5 g of fresh sludge from a chemical wastewater pool, vortex and mix thoroughly in 10 mL of sterile water. After standing, take 1 mL of the upper suspension and inoculate it into enrichment medium 1 at a 2% inoculation rate. Culture it at 30°C, 220 rpm for 5 days to obtain enrichment fermentation broth 1. This method was used to increase the methanol content in the culture medium. The enrichment fermentation broth 1 was transferred to enrichment medium 2 and enrichment medium 3 for 5 days at 30°C, 220 rpm. After each transfer, the fermentation broth that darkened and turned red was selected for centrifugation to obtain the supernatant. The PQQ titer in the fermentation broth was detected by high performance liquid chromatography.

[0053] (2) The enriched fermentation broth with the highest PQQ titer was selected and gradiently diluted, then spread on enrichment plates and cultured at 30°C for 5 days. Single colonies with good growth and clear edges were selected and transferred to screening medium and cultured at 30°C and 220 rpm for 6 days. The methanol content was measured on the second and fourth days during the culture process. Methanol was added when the methanol content was low. After the culture was completed, the PQQ titer in the fermentation broth was determined by HPLC. The strain with the highest titer was selected and recorded as HX-SX-81 for mutagenesis treatment.

[0054] The culture medium components of this embodiment include: 1. Basic culture medium: (NH4)2SO42.0 g / L, KH2PO41.5 g / L, Na2HPO4·12H2O 9.0 g / L, MgSO4·7H2O 2.0 g / L, CaCO31.0 g / L, trace element solution 0.7 mL / L; 2. Enrichment medium 1 4 g / L methanol was added to the basal medium as a carbon source.

[0055] 3. Enrichment medium 2 6 g / L methanol was added as a carbon source to the basal medium.

[0056] 4. Enrichment medium 3 8 g / L methanol was added to the basal medium as a carbon source.

[0057] 5. Enrichment Plate 20 g / L agar powder and 10 g / L methanol were added to the basal medium as a carbon source.

[0058] 6. Screening medium 20 g / L methanol was added to the basal medium as a carbon source; the trace element solution composition was ZnSO4·7H2O15.0 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, CaCl222.65 g / L, (NH4)6Mo7O 24 ·4H2O 0.05 g / L, H3BO30.05 g / L, KI 0.05 g / L.

[0059] Example 2 Mutation breeding of high-yield PQQ bacteria This embodiment provides a method for breeding high-yield PQQ bacteria by physical and chemical combined mutagenesis using high concentration methanol as an antagonistic factor, and the specific steps are as follows: (1) The strain HX-SX-81 obtained in Example 1 was used as the original strain and inoculated into the screening medium and cultured until the seed liquid OD 600 The value is 1.6, 5 mL of bacterial suspension is centrifuged to remove the supernatant, washed twice with PBS buffer and resuspended, 2 mL of bacterial suspension is placed under a sterile plate, placed 30 cm from a preheated 25W UV lamp, and sterile magnetic beads are stirred and irradiated at 120 rpm under a magnetic stirrer for 20 s to 60 s (e.g. 20 s, 30 s, 40 s, 50 s, 60 s), and nitrosoguanidine is added to the UV-irradiated bacterial suspension at an amount of 1 mg / L to 5 mg / L (e.g. 1 mg / L, 2 mg / L, 3 mg / L), mixed and cultured at 30°C in the dark for 60 min, centrifuged for 10 min to remove the supernatant, washed twice with PBS and resuspended. The bacterial suspension treated with UV-nitrosoguanidine composite mutagenesis and the bacterial suspension not treated with mutagenesis are appropriately diluted and spread on the screening plate, and cultured at 30°C in the dark for 8 days.

[0060] (2) After the culture was completed, the lethality was calculated based on the number of colonies on the untreated plates and the plates with different mutagenic doses. Single colonies with good growth and clear colony edges on the plates with a lethality of 90% were selected and inoculated into the screening medium. The culture was carried out at 30°C and 220 rpm for 6 days. Methanol was added every two days according to the residual methanol content in the culture medium during the culture process. After the culture was completed, the PQQ titer in the fermentation broth was determined by HPLC. Strains with higher PQQ titers were selected for a new round of compound mutagenesis, and the process was repeated three times.

[0061] (3) After the three rounds of compound mutagenesis, the bacterial suspension was spread on the screening plate 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 at 30°C and 220 rpm for 62 h until the seed liquid OD reached 600The value was 3.5, and the culture medium was inoculated with 1.5% inoculation rate at 30℃ and 220 rpm for 6 days. Methanol was added every 2 days during the culture process to a methanol concentration of 5 g / L. After the culture was completed, the PQQ titer in the fermentation broth was detected by HPLC.

[0062] A high-yield PQQ starting strain numbered HX-YB-32 was screened using the above steps. After 6 days of shake flask fermentation, the titer reached 127.2 mg / L.

[0063] The screening plate and screening medium described in this example are the same as the enrichment plate and screening medium described in Example 1, and the fermentation shake flask medium is the basal medium described in Example 1 plus 5 g / L methanol as the sole carbon source.

[0064] Example 3 Strain identification and preservation The mutant strain HX-YB-32 screened in Example 2 was subjected to physiological and biochemical index detection, morphological observation and species identification.

[0065] The results showed that the strain was an aerobic bacterium, with negative results for methyl red, VP and starch hydrolysis tests, positive for oxidase, able to reduce nitrite, no production of indole and hydrogen sulfide, able to utilize methanol, arabinose and trehalose, sensitive to tetracycline, but insensitive to ampicillin, kanamycin and chloramphenicol, with the optimal growth temperature of 28-32°C and pH 6-8. The colonies were small and milky white, with a smooth surface and Gram staining negative.

[0066] The DNA of the mutagenic strain HX-YB-32 was used as a template and 16S rDNA universal primers were used for PCR amplification. The amplified product was sent to Qingdao Weilai Biotechnology for sequencing, and the 16S rDNA sequence of the strain was obtained as follows (SEQ ID No. 1): The 16S rDNA sequence of this strain was compared with that of H. denitrificans strain ATCC 51888, H. denitrificans strain DSM 1869 was more than 99%. H. denitrificans Divided into the same branch (such as Figure 1 Therefore, the strain was identified and named H. denitrificans HXRD10-32.

[0067] The strain was deposited in the China Center for Type Culture Collection (CCTCC) on November 21, 2024, with the address being: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the deposit number is: CCTCC NO: M 20242618.

[0068] Example 4 Production of PQQ by Denitrifying Mycelial Microbes Fermentation This embodiment utilizes H. denitrificans HXRD10-32 is fermented to produce PQQ, comprising the following steps: (1) Culture medium ① Seed culture medium The composition of the seed culture medium is (NH4)2SO42 g / L, KH2PO41.5 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, trace element D solution 0.7 mL / L. Make up to 100 mL in a 500 mL conical flask, sterilize at 121 ℃ for 20 min, and add methanol to 10 g / L.

[0069] ② Fermentation medium The fermentation medium composition is (NH4)2SO42 g / L, KH2PO41.5 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, trace element D solution 0.7 mL / L. The volume is adjusted to 9 L and placed in a 15 L fermentation tank. After sterilization at 121℃ for 20 minutes, methanol is added to 5 g / L.

[0070] In the above seed culture medium and fermentation medium, the composition of trace element A liquid 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 B liquid is NaCl 1.5 g / L, CoCl·6H2O 0.05 g / L, CaCl222.65 g / L; the composition of trace element C liquid is (NH4)6Mo7O 24 4H2O 0.05 g / L, H3BO3 0.05 g / L; the composition of trace element D liquid is KI 0.05 g / L.

[0071] (2) Seed cultivation Pick H. denitrificans The HXRD10-328 glycerol bacteria were thawed at room temperature, and the glycerol bacteria solution was inoculated into the seed culture medium at 0.6%. After inoculation, the glycerol bacteria were cultured on a shaking table at 30 °C and 200 rpm until the OD 600 The value 3.5 is for future use.

[0072] (3) Fermentation in 15L fermenter After the cultured seeds are confirmed to be free of foreign bacteria under microscopic examination, they are inoculated at a 10% inoculum into a 15 L fermentation tank containing fermentation medium for fermentation.

[0073] (4) 15 L tank fermentation process control During the fermentation cycle, the fermentation tank liquid volume was 9 L, the culture temperature was 30°C, the initial stirring speed was 150 rpm, the air flow rate was 1.0 VVM, the ammonia water self-controlled pH value was 6.8, and the culture period was 240 h.

[0074] (5) 15L tank fermentation dissolved oxygen control The dissolved oxygen during the entire fermentation cycle is controlled at more than 30% through stirring linkage.

[0075] (6) 15 L tank methanol fermentation control The fermentation process was controlled by adding methanol. At the initial stage of fermentation, 50% methanol was added to the fermentation medium to achieve a methanol concentration of 5.0 g / L, followed by inoculation. Methanol was continuously consumed as the bacteria grew. The methanol concentration in the fermentation broth was monitored regularly during the fermentation cycle, and the methanol concentration in the fermentation broth was controlled to be 3-5 g / L by adjusting the flow rate of 50% methanol.

[0076] (7) Trace element control in 15 L tank fermentation The fermentation process was controlled by adding trace element liquid. At the beginning of the fermentation, the initial concentration of trace elements A, B, C, and D in the culture medium was 1.0 mL / L. 600Start growing, during fermentation OD 600 When the temperature is higher than 60, add trace element solution once. The supplementary amount of trace element A, B, C, and D solution is 0.5 mL / L.

[0077] (8) 15 L tank fermentation effect test The whole fermentation cycle had an impact on methanol content and OD 600 The titer of PQQ was tested, the methanol content was determined by ethanol flow addition detector, and the OD 600 The PQQ titer was determined by spectrophotometer, and the PQQ titer was determined by high performance liquid chromatography. The conditions for high performance liquid chromatography determination were as follows: chromatographic column TCI Kaseisorb LC ODS 2000, injection volume 20 μL, column temperature 30 ℃, flow rate 1.0 mL / min, mobile phase A was 10 mmol / L potassium dihydrogen phosphate-15 mmol / L tetrabutylammonium bromide mixed solution (pH 7.4), mobile phase B was acetonitrile, gradient elution was used, and the gradient elution program was: 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, PQQ peak time was about 14.55min (such as Figure 2 Fermentation cycle OD 600 and PQQ titer changes as Figure 3 shown.

[0078] The titer of PQQ in this example is 3.25 g / L, OD 600 It is 113.20.

[0079] Comparison Example 1 The difference between this comparative example and Example 4 is that the initial addition amount of the trace elements A, B, C, and D liquid in the fermentation medium is 0.5 mL / L, and the rest is the same as Example 4.

[0080] The fermentation cycle had an impact on methanol content and OD 600 The titer of PQQ was tested. During the fermentation process, 50% methanol after sterile filtration was added to supplement the carbon source. 600 The results of the whole process of testing PQQ potency are as follows Figure 4 As shown in the figure, after 240 h of fermentation, the PQQ titer was 2.35 g / L and the OD 600 It is 73.10.

[0081] Comparative Example 2 The difference between this comparative example and Example 4 is that the initial addition amount of the trace elements A, B, C, and D liquid in the fermentation medium is 1.3 mL / L, and the rest is the same as Example 4.

[0082] The fermentation cycle had an impact on methanol content and OD 600 The titer of PQQ was tested. During the fermentation process, 50% methanol after sterile filtration was added to supplement the carbon source. 600 The results of the whole process of testing PQQ potency are as follows Figure 5 As shown in the figure, after 240 h of fermentation, the PQQ titer was 2.47 g / L and the OD 600 It is 127.60.

[0083] Comparative Example 3 The difference between this comparative example and Example 4 is that the initial addition amount of the trace elements A, B, C, and D liquid in the fermentation medium is 1.6 mL / L, and the rest is the same as Example 4.

[0084] The fermentation cycle had an impact on methanol content and OD 600 The titer of PQQ was tested. During the fermentation process, 50% methanol after sterile filtration was added to supplement the carbon source. 600 The results of the whole process of testing PQQ potency are as follows Figure 6 As shown in the figure, after 240 h of fermentation, the PQQ titer was 2.13 g / L and the OD 600 It is 139.60.

[0085] Comparative Example 4 The difference between this comparative example and Example 4 is that the initial addition amount of the trace elements A, B, C, and D liquid in the fermentation medium is 2.0 mL / L, and the rest is the same as Example 4.

[0086] The fermentation cycle had an impact on methanol content and OD 600 The titer of PQQ was tested. During the fermentation process, 50% methanol after sterile filtration was added to supplement the carbon source. 600 The results of the whole process of testing PQQ potency are as follows Figure 7 As shown in the figure, after 240 h of fermentation, the PQQ titer was 1.98 g / L and the OD 600 It is 126.60.

[0087] Comparative Example 5 The difference between this comparative example and Example 4 is that except for the initial addition of trace element liquid to the fermentation medium, no trace element liquid is added during the fermentation cycle, and the rest is the same as Example 4.

[0088] The fermentation cycle had an impact on methanol content and OD 600 The titer of PQQ was tested. During the fermentation process, 50% methanol after sterile filtration was added to supplement the carbon source. 600 The results of the whole process of testing PQQ potency are as follows Figure 8 As shown in the figure, after 240 h of fermentation, the PQQ titer was 2.98 g / L and the OD600 It is 109.20.

[0089] Comparative Example 6 The difference between this comparative example and Example 4 is that in addition to the initial addition of trace element liquid to the fermentation medium, trace element liquid was added once during the fermentation process when OD600>60, and the addition amount of trace element A, B, C, and D liquid was 1.0 mL / L.

[0090] The fermentation cycle had an impact on methanol content and OD 600 The titer of PQQ was tested. During the fermentation process, 50% methanol after sterile filtration was added to supplement the carbon source. 600 The results of the whole process of testing PQQ potency are as follows Fig. 9 As shown in the figure, after 240 h of fermentation, the PQQ titer was 2.77 and the OD 600 It is 107.40.

[0091] Compared with Example 4, the fermentation effects of Comparative Examples 1 to 6 are all lower than the final fermentation effects, as shown in Table 1.

[0092] Table 1 Effects of different trace element additions on PQQ potency

[0093] By comparing Comparative Example 1 and Example 4, it can be seen that the initial addition of trace elements will seriously affect the growth of bacteria. The initial addition of Comparative Example 1 is too low to affect the growth and reproduction of bacteria. 600 Significantly lower than Example 4.

[0094] 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 the initial addition of trace elements is too high, resulting in a seriously low potency.

[0095] By comparing Comparative Examples 5 and 6 with Example 4, it can be seen that supplementing trace elements once in an appropriate amount during the fermentation process is beneficial to product synthesis, but excessive supplementation will also affect product synthesis.

[0096] The above results show that by optimizing the initial trace element content in the fermentation medium and by adding appropriate amounts of trace elements during the fermentation cycle, the H. denitrificans HXRD10-328 bacterial growth metabolism and can significantly increase OD 600 Value and PQQ titer.

[0097] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teaching content of this application, those skilled in the art can make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A denitrifying hyphae microorganism HXRD10-32 ( Hyphomicrobium denitrificans HXRD10-32), deposited in China Center for Type Culture Collection, with the deposit number being CCTCC NO: M 20242618, and the deposit date being November 21, 2024.

2. A bacterial agent, characterized in that It comprises the denitrifying hyphae microbacterium HXRD10-32 described in claim 1.

3. A fermentation broth, characterized in that: It comprises the denitrifying hyphae microbacterium HXRD10-32 described in claim 1.

4. Use of the denitrifying hyphae microbacterium HXRD10-32 according to claim 1, the bacterial agent according to claim 2 or the fermentation liquid according to claim 3, characterized in that: Used for preparing pyrroloquinoline quinone; or, used for preparing one or more of human medicine, veterinary medicine, health care products, food, beverage, cosmetics, additives and feed containing pyrroloquinoline quinone.

5. A method for producing pyrroloquinoline quinone, characterized in that: The method comprises the following steps: fermenting and culturing the denitrifying hyphae microbacterium HXRD10-32 according to claim 1 or the bacterial agent according to claim 2 to obtain the pyrroloquinoline quinone.

6. The method according to claim 5, characterized in that The denitrifying hyphae microbacterium HXRD10-32 or the bacterial agent is fermented in a fermentation medium containing an assimilable carbon source.

7. The method according to claim 6, characterized in that The assimilable carbon source is methanol, or methylamine, or other carbon sources in addition to methanol, and the other carbon sources are at least selected from methylamine, ethanol, glycerol, formic acid, acetic acid, mannitol, starch, maltodextrin, glucose, sucrose, lactose, maltose, industrial molasses, soybean oil, sorbitol, or any combination thereof.

8. The method according to claim 6, characterized in that The fermentation medium also 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.

9. The method according to any one of claims 6 to 8, characterized in that: Before fermentation, a trace element liquid is added to the fermentation medium, and the trace element liquid is added at least once during the fermentation process. The solutes in the trace element liquid include ZnSO4, MnSO4, CuSO4, FeSO4, NaCl, CoCl, CaCl2, (NH4)6Mo7O 24 , H3BO3 and KI.

10. The method according to any one of claims 6 to 8, characterized in that: The fermentation process included methanol feed control.

Images