Method for high-yield fermentation of pyrroloquinoline quinine by using methylobacterium extorquens

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for high-yield fermentation of pyrroloquinoline quinone by utilizing methylobacterium extorquens. Methylobacterium extorquens is fermented and cultured through a specific lignocellulose hydrolysate culture medium, a methanol dehydrogenase inhibitor is added to ferment the methylobacterium extorquens, lysozyme is added to release intracellular PQQ, and the yield of the PQQ is increased by 5-10 times and can reach 1973.0 mg / L. In addition, lignocellulose from agriculture and forestry biomass waste is used as the raw material, the application range of the renewable plant-based biomass raw material is greatly expanded, and the raw material is wide in source, low in cost, simple in preparation method and suitable for industrial large-scale production.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for highly productive fermentation of pyrroloquinoline quinone. Background Art

[0002] Pyrroloquinoline quinone (hereinafter referred to as PQQ) is a quinone compound synthesized by methylotrophic bacteria and belongs to the third class of redox enzyme cofactors after nicotinamide and riboflavin. PQQ exhibits significant biological functions, especially in the prevention and treatment of certain diseases. This compound is widely distributed in prokaryotes, the plant kingdom, and even mammals. It not only serves as a key cofactor for various enzymes, playing a crucial role in electron, proton, and chemical group transfer during enzyme catalysis, but also shows a stimulatory effect on microbial growth, promoting pollen germination and overall growth and development of plants. In recent years, in-depth research on the distribution, biosynthesis mechanism, structure, function, and biological properties of PQQ, as well as research on its development as a new drug, has attracted extensive attention internationally. The application potential of PQQ in multiple fields such as food, light industry, agriculture, and medicine cannot be ignored.

[0003] The biosynthesis of PQQ mainly depends on the metabolic activities of microorganisms. In recent years, with the rapid development of bioinformatics and genetic engineering technologies, the biosynthesis pathway of PQQ and its key enzyme catalytic mechanism have been widely studied. Currently, known organisms capable of producing pyrroloquinoline quinone include Acinetobacter calcoaceticus, Klebsiella pneumoniae, Gluconobacter oxydans, Methylobacter flagellatus, Methylobacterium extorquens, Hyphomicrobium, Methylophilus, etc. Patent 202410192595.9 discloses a mutagenized strain of Methylobacterium extorquens for fermentative production of pyrroloquinoline quinone, its breeding method and application. By ultraviolet mutagenesis to breed a high-yield pyrroloquinoline quinone strain, a relatively optimal positive mutant Methylobacterium extorquens B70 was obtained, with a fermentation production of PQQ of 157.3 mg / L. The fermentation titer is relatively low, far from meeting the industrialization requirements, and the mutagenized strain may have genetic instability and may lose its high-yield characteristics during subculture, resulting in unstable production. Patent 202110967604.3 discloses a method for regulating the production of pyrroloquinoline quinone by methylotrophic bacteria using redox potential. In the system of fermentative production of pyrroloquinoline quinone by methylotrophic bacteria, a redox potential electrode is introduced, and the redox potential change during the fermentation production stage is adjusted by controlling the aeration volume and stirring speed in the system, thereby ensuring cell growth and the accumulation of pyrroloquinoline quinone, shortening the fermentation time, and simultaneously increasing the yield and productivity of pyrroloquinoline quinone and the stability of the fermentation system. However, the method of regulating the production of pyrroloquinoline quinone by methylotrophic bacteria using redox potential has a high cost and is not suitable for large-scale industrial production.

[0004] With the growth of market demand, the breeding of high-yield strains and the development of their efficient purification processes have become the primary issues in the industrial production of PQQ. Therefore, there is an urgent need to develop an efficient microbial fermentation production process that can simplify the preparation method, reduce costs, and meet the requirements of large-scale production, providing technical support for industrial production. Summary of the Invention

[0005] To solve the above problems, the present invention proposes a method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens.

[0006] The technical solution of the present invention is realized as follows:

[0007] On the one hand, the present application provides a method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens: inoculate Methylobacterium extorquens into a culture medium, add Vitreoscilla hemoglobin and stir-ferment to obtain a mixed solution A, and add lysozyme to the mixed solution A to obtain a fermentation broth rich in PQQ.

[0008] Preferably, the mass ratio of the above Vitreoscilla hemoglobin to the culture medium is 4 - 10:10000; the amount of lysozyme added to the mixed solution A is 0.2 - 0.7 parts by mass.

[0009] Preferably, the components of the above culture medium include 900 - 1000 parts by mass of lignocellulase hydrolyzate, 2 - 5 parts by mass of corn steep liquor, 2 - 6 parts by mass of K2HPO4, 3 - 8 parts by mass of NaH2PO4, 0.05 - 3 parts by mass of MgSO4·7H2O, 1 - 9 parts by mass of ammonium sulfate, 1 - 2 parts by mass of trace element A solution, and 0.5 - 1 part by mass of trace element B solution.

[0010] Preferably, the above trace element A solution includes 1000000 - 1500000 parts by mass of water, 5 - 20 parts by mass of CaCl2·2H2O, 10 - 20 parts by mass of ZnSO4·7H2O, 2 - 4 parts by mass of MnCl2, 0.1 - 1 part by mass of CuSO4·5H2O, and 1 - 3 parts by mass of NaCl.

[0011] Preferably, the above trace element B solution includes 1000000 - 1500000 parts by mass of water, 10 - 15 parts by mass of (NH4)6Mo7O 24 ·4H2O, 10 - 15 parts by mass of KI, 10 - 15 parts by mass of CoCl2·6H2O, and 10 - 15 parts by mass of H3BO3.

[0012] Furthermore, the specific steps of the above method are as follows:

[0013] Step 1: Prepare lignocellulase hydrolyzate:

[0014] Add 80 - 150 g of lignocellulose to 1 L of water, add complex cellulase and react to obtain lignocellulase hydrolyzate;

[0015] Step 2: Prepare trace element A solution:

[0016] Add 1,000,000 - 1,500,000 parts of water, 5 - 20 parts of CaCl₂·2H₂O, 10 - 20 parts of ZnSO₄·7H₂O, 2 - 4 parts of MnCl₂, 0.1 - 1 part of CuSO₄·5H₂O, and 1 - 3 parts of NaCl into a container, and stir evenly to obtain trace element A solution;

[0017] Step 3: Prepare trace element B solution:

[0018] Add 1,000,000 - 1,500,000 parts of water, 10 - 15 parts of (NH₄)₆Mo₇O 24 ·4H₂O, 10 - 15 parts of KI, 10 - 15 parts of CoCl₂·6H₂O, and 10 - 15 parts of H₃BO₃ into a container, and stir evenly to obtain trace element B solution;

[0019] Step 4: Prepare culture medium solution:

[0020] Add 900 - 1000 parts of lignocellulase hydrolyzate, 2 - 5 parts of corn steep liquor, 2 - 6 parts of K₂HPO₄, 3 - 8 parts of NaH₂PO₄, 0.05 - 3 parts of MgSO₄·7H₂O, 6 - 9 parts of ammonium sulfate, 1 - 2 parts of trace element A solution, and 0.5 - 1 part of trace element B solution into the culture medium to obtain culture medium solution;

[0021] Step 5: After activating Methylobacterium extorquens, inoculate it into the culture medium, and add Vitreoscilla hemoglobin, and stir and ferment to obtain mixture A;

[0022] Step 6: Add 0.2 - 0.7 part of lysozyme to mixture A to obtain a fermentation broth containing PQQ.

[0023] Furthermore, the activity of cellulase in the above lignocellulase hydrolyzate is 40 - 50 FPU / g, the pH of the phosphate buffer solution in the reaction is 7.2 - 7.8, and the reaction temperature is 20 - 30 °C.

[0024] Furthermore, the above steps also include adding mixture C composed of phenoxyquinoline, methanol, formamide, ferrous sulfate, calcium chloride, and Triton X - 100 to the culture medium after stirring and fermenting for 4 - 6 h; the mass ratio of mixture C to mixture A is 6 - 7:10.

[0025] Further, the mass parts of the above mixture C are 2 - 10 parts of phenoxyquinoline, 1000 - 5000 parts of methanol, 100 - 500 parts of formamide, 20 - 90 parts of ferrous sulfate, 20 - 60 parts of calcium chloride, 1000 - 2000 parts of Triton X-100, and 10000 - 20000 parts of water per 100,000 parts of the fermentation broth; wherein phenoxyquinoline is a PQQ-dependent methanol dehydrogenase inhibitor, methanol is a well-known carbon source in the art, formamide is a well-known nitrogen source in the art, ferrous sulfate and calcium chloride are well-known cofactors in the art, and Triton X-100 is a well-known cell membrane permeabilizer in the art.

[0026] Preferably, the time of the above stirring fermentation is 60 - 80 h, and the temperature is 20 - 35 °C.

[0027] In the second aspect, the application of the above method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens in the industrial production of pyrroloquinoline quinone.

[0028] The present invention has the following beneficial effects:

[0029] In this application, Methylobacterium extorquens is fermented and cultured through a specific lignocellulase hydrolyzate medium, and phenoxyquinoline with an inhibitory effect on PQQ-dependent methanol dehydrogenase, carbon source methanol, nitrogen source formamide, cofactors ferrous sulfate and calcium chloride, and cell membrane permeabilizer Triton X-100 are added to the medium to optimize the fermentation process, so that the PQQ yield is increased by 5 - 10 times, and the yield can reach 1973.0 mg / L; the PQQ purified product is identified by mass spectrometry and ultraviolet spectroscopy, and is consistent with the reported molecular weight and ultraviolet spectrum of PQQ. Figure 1 In addition, this application uses lignocellulose derived from agricultural and forestry biomass waste as a raw material to produce high-value-added pyrroloquinoline quinone, greatly expanding the application range of renewable plant-based biomass raw materials. The raw materials of the present invention are widely sourced, low in cost, and the preparation method is simple, suitable for large-scale industrial production. Description of the Drawings

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0031] Figure 1 It is the schematic diagram of the inhibitory effect of phenoxyquinoline on PQQ-dependent methanol dehydrogenase.

[0032] Figure 2 It is the PQQ detection standard curve graph in the present invention.

[0033] Figure 3 HPLC chromatogram of PQQ in the fermentation broth of Example 3

[0034] Figure 4 HPLC chromatogram of PQQ standard

[0035] Figure 5 Mass spectrum of PQQ in the fermentation broth of Example 3

[0036] Figure 6 UV spectrum of PQQ in the fermentation broth of Example 3 Detailed implementation manners

[0037] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0038] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0039] The present application provides a method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens: inoculate Methylobacterium extorquens into a culture medium, add Vitreoscilla hemoglobin, and then add a mixture C composed of phenoxyquinoline, methanol, formamide, ferrous sulfate, calcium chloride and Triton X-100 to the culture medium to obtain a fermentation broth; add lysozyme to the fermentation broth to promote the release of intracellular pyrroloquinoline quinone, and obtain a fermentation broth rich in PQQ. The schematic diagram of the inhibitory effect of phenoxyquinoline on PQQ-dependent methanol dehydrogenase is as Figure 1 shown. This figure shows the molecular conformation when phenoxyquinoline binds to PQQ (pyrroloquinoline quinone)-dependent methanol dehydrogenase. The figure shows that phenoxyquinoline, as an inhibitor, binds to the key active site of the enzyme, thereby blocking the binding of the enzyme to the substrate (such as methanol), and further inhibiting the activity of the enzyme.

[0040] Methylobacterium extorquens ATCC 27329, Methylobacterium extorquens DSM1340 and Methylobacterium extorquens ATCC 43645 used in the present application are all purchased from Beijing BioWin Biotechnology Co., Ltd.

[0041] Example 1

[0042] A method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens, comprising the following steps:

[0043] Step 1: Prepare lignocellulase hydrolysate:

[0044] Add 150 g of lignocellulose to 1 L of water, add complex cellulase, and obtain lignocellulose enzyme hydrolysate;

[0045] Among them, the substrate dry weight of the cellulase is 50 FPU / g; the pH of the phosphoric acid buffer solution used is 7.2, and the reaction temperature is 30 °C.

[0046] Step 2: Prepare trace element A solution:

[0047] Add 1,500,000 parts of water, 20 parts of CaCl₂·2H₂O, 20 parts of ZnSO₄·7H₂O, 4 parts of MnCl₂, 1 part of CuSO₄·5H₂O, and 3 parts of NaCl to a container, and stir evenly to obtain trace element A solution.

[0048] Step 3: Prepare trace element B solution:

[0049] Add 1,500,000 parts of water, 15 parts of (NH₄)₆Mo₇O 24 ·4H₂O, 15 parts of KI, 15 parts of CoCl₂·6H₂O, and 15 parts of H₃BO₃ to a container, and stir evenly to obtain trace element B solution.

[0050] Step 4: Prepare culture medium solution:

[0051] Add 1000 parts of lignocellulose enzyme hydrolysate, 5 parts of corn steep liquor, 6 parts of K₂HPO₄, 8 parts of NaH₂PO₄, 3 parts of MgSO₄·7H₂O, 9 parts of ammonium sulfate, 2 parts of trace element A solution, and 1 part of trace element B solution to the culture medium to obtain culture medium solution.

[0052] After activating Methylobacterium extorquens ATCC 27329, inoculate it into the culture medium, and add Vitreoscilla hemoglobin and stir for high-density fermentation. After 80 h, obtain mixture A; among them, the fermentation temperature is 35 °C; the rotation speed is 200 r / min;

[0053] After fermenting for 6 h, add mixture C composed of phenoxyquinoline, methanol, formamide, ferrous sulfate, calcium chloride, and Triton X-100 to the culture medium, and the mass ratio of mixture C to mixture A is 7:10.

[0054] Mixture C can be added using any common feeding method in the art. Here, taking the common batch feeding method in the art as an example, after 6 hours of fermentation, 1% of the mass of the fermentation broth of Mixture C is added as a supplement every 120 minutes. For every 100,000 parts of the fermentation broth, 10 parts of phenoxyquinoline, 5000 parts of methanol, 500 parts of formamide, 90 parts of ferrous sulfate, 60 parts of calcium chloride, 2000 parts of Triton X-100, and 20,000 parts of water are added. Among them, phenoxyquinoline is used as a PQQ-dependent methanol dehydrogenase inhibitor, methanol is used as a carbon source, formamide is used as a nitrogen source, ferrous sulfate and calcium chloride are used as cofactors, and Triton X-100 is used as a cell membrane permeabilizer; the batch feeding method is a common feeding method in the art to maintain the dynamic balance of nutrients in the fermentation broth, which can avoid substrate inhibition and thus significantly increase the yield;

[0055] Among them, the mass ratio of Vitreoscilla hemoglobin to the culture medium solution is 10:10000.

[0056] Step Six: Add 0.7 parts by mass of lysozyme to the mixed solution A to promote the release of intracellular pyrroloquinoline quinone, obtaining a fermentation broth containing PQQ.

[0057] Step Seven: Measure the content of PQQ in the fermentation broth.

[0058] Example 2

[0059] A method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens, the steps are as follows:

[0060] Step One: Prepare a lignocellulase hydrolysate:

[0061] Add 80 g of lignocellulose to 1 L of water, and add a composite cellulase to obtain a lignocellulase hydrolysate;

[0062] Among them, the substrate dry weight of the cellulase is 40 FPU / g; the pH of the phosphoric acid buffer solution used is 7.8, and the reaction temperature is 20 °C.

[0063] Step Two: Prepare a trace element A solution:

[0064] Add 1,000,000 parts of water, 5 parts of CaCl2·2H2O, 10 parts of ZnSO4·7H2O, 2 parts of MnCl2, 0.1 part of CuSO4·5H2O, and 1 part of NaCl to a container, and stir evenly to obtain a trace element A solution.

[0065] Step Three: Prepare a trace element B solution:

[0066] Add 1,000,000 parts of water, 10 parts of (NH4)6Mo7O 24· 4H2O, 10 parts of KI, 10 parts of CoCl2·6H2O, 10 parts of H3BO3, and after stirring evenly, a trace element B solution is obtained.

[0067] Step 4: Prepare the culture medium solution:

[0068] Add 900 parts of lignocellulase hydrolyzate, 2 parts of corn steep liquor, 2 parts of K2HPO4, 3 parts of NaH2PO4, 0.05 part of MgSO4·7H2O, 1 part of ammonium sulfate, 1 part of trace element A solution, and 0.5 part of trace element B solution to the culture medium to obtain the culture medium solution.

[0069] Step 5: After activating Methylobacillus flagellatus ATCC 27329, inoculate it into the culture medium, and add Vitreoscilla hemoglobin and stir for high-density fermentation. After 60 h, a mixed solution A is obtained; wherein the fermentation temperature is 20 °C; the rotation speed is 100 r / min;

[0070] After 4 h of fermentation, add a mixture C composed of phenoxyquinoline, methanol, formamide, ferrous sulfate, calcium chloride, and Triton X-100 to the culture medium, and the mass ratio of the mixture C to the mixed solution A is 6:10.

[0071] The mixture C can be selected by any common feeding method in the art. Here, taking the common intermittent feeding method in the art as an example, after 4 h of fermentation, add 2% of the mixture C of the fermentation broth mass to the culture medium every 100 min. Add 2 parts of phenoxyquinoline, 1000 parts of methanol, 100 parts of formamide, 20 parts of ferrous sulfate, 20 parts of calcium chloride, 1000 parts of Triton X-100, and 10000 parts of water to every 100000 parts of the fermentation broth. Among them, phenoxyquinoline is used as a PQQ-dependent methanol dehydrogenase inhibitor, methanol is used as a carbon source, formamide is used as a nitrogen source, ferrous sulfate and calcium chloride are used as cofactors, and Triton X-100 is used as a cell membrane permeabilizer; the intermittent feeding is a common feeding method in the art to maintain the dynamic balance of nutrients in the fermentation broth, which can avoid substrate inhibition and thus significantly increase the yield;

[0072] Among them, the mass ratio of Vitreoscilla hemoglobin to the culture medium solution is 4:10000.

[0073] Step 6: Add 0.2 parts by mass of lysozyme to the mixed solution A to promote the release of intracellular pyrroloquinoline quinone, and obtain a fermentation broth containing PQQ.

[0074] Step 7: Measure the content of PQQ in the fermentation broth.

[0075] Example 3

[0076] A method for high-yield fermentation of pyrroloquinoline quinone using Methylobacillus flagellatus, the steps are as follows:

[0077] Step 1: Prepare the lignocellulose enzyme hydrolysate:

[0078] Add 100 g of lignocellulose to 1 L of water, and add a complex cellulase to obtain the lignocellulose enzyme hydrolysate;

[0079] Among them, the substrate dry weight of the cellulase is 45 FPU / g; the pH of the used phosphate buffer solution is 7.5, and the reaction temperature is 28 °C.

[0080] Step 2: Prepare the trace element A solution:

[0081] Add 1,000,000 parts of water, 10 parts of CaCl₂·2H₂O, 15 parts of ZnSO₄·7H₂O, 3 parts of MnCl₂, 0.3 parts of CuSO₄·5H₂O, and 1 part of NaCl to a container, and stir evenly to obtain the trace element A solution.

[0082] Step 3: Prepare the trace element B solution:

[0083] Add 1,000,000 parts of water, 10 parts of (NH₄)₆Mo₇O 24 ·4H₂O, 15 parts of KI, 10 parts of CoCl₂·6H₂O, and 10 parts of H₃BO₃ to a container, and stir evenly to obtain the trace element B solution.

[0084] Step 4: Prepare the culture medium solution:

[0085] Add 950 parts of the lignocellulose enzyme hydrolysate, 3 parts of corn steep liquor, 4 parts of K₂HPO₄, 5 parts of NaH₂PO₄, 1 part of MgSO₄·7H₂O, 5 parts of ammonium sulfate, 1.5 parts of the trace element A solution, and 0.6 parts of the trace element B solution to the culture medium to obtain the culture medium solution.

[0086] Step 5: After activating Methylobacterium extorquens ATCC 27329, inoculate it into the culture medium, and add Vitreoscilla hemoglobin and stir for high-density fermentation. After 70 h, obtain the mixture A; among them, the fermentation temperature is 30 °C; the rotation speed is 150 r / min;

[0087] After 5 h of fermentation, add the mixture C composed of quinoxaline, methanol, formamide, ferrous sulfate, calcium chloride, and Triton X-100 to the culture medium. The mass ratio of the mixture C to the mixture A is 6.5:10;

[0088] Mixture C can be added by any common feeding method in the art. Here, taking the common batch feeding method in the art as an example, after 5 hours of fermentation, 5% of mixture C based on the mass of the fermentation broth is added to the culture medium every 120 minutes. For every 100,000 parts of the fermentation broth, 6 parts of phenoxyquinoline, 3000 parts of methanol, 300 parts of formamide, 50 parts of ferrous sulfate, 50 parts of calcium chloride, 1500 parts of Triton X-100, and 15000 parts of water are added. Among them, phenoxyquinoline is used as a PQQ-dependent methanol dehydrogenase inhibitor, methanol is used as a carbon source, formamide is used as a nitrogen source, ferrous sulfate and calcium chloride are used as cofactors, and Triton X-100 is used as a cell membrane permeabilizer; the batch feeding is a common feeding method in the art to maintain the dynamic balance of nutrients in the fermentation broth, which can avoid substrate inhibition and thus significantly increase the yield;

[0089] Among them, the mass ratio of Vitreoscilla hemoglobin to the culture medium solution is 5:10000.

[0090] Step Six: Add 0.5 parts by mass of lysozyme to the mixed solution A to promote the release of intracellular pyrroloquinoline quinone, and obtain a fermentation broth containing PQQ.

[0091] Step Seven: Measure the content of PQQ in the fermentation broth.

[0092] Example 4

[0093] A method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens, the steps are as follows:

[0094] Step One: Prepare lignocellulose enzyme hydrolysate:

[0095] Add 100 g of lignocellulose to 1 L of water, and add complex cellulase to obtain lignocellulose enzyme hydrolysate;

[0096] Among them, the substrate dry weight of the cellulase is 45 FPU / g; the pH of the used phosphate buffer solution is 7.5, and the reaction temperature is 28 °C.

[0097] Step Two: Prepare trace element A solution:

[0098] Add 1000000 parts of water, 10 parts of CaCl2·2H2O, 15 parts of ZnSO4·7H2O, 3 parts of MnCl2, 0.5 parts of CuSO4·5H2O, and 1 part of NaCl to a container, and stir evenly to obtain trace element A solution.

[0099] Step Three: Prepare trace element B solution:

[0100] Add 1000000 parts of water, 10 parts of (NH4)6Mo7O 24· 4H2O, 15 parts of KI, 15 parts of CoCl2·6H2O, 10 parts of H3BO3, and after stirring evenly, a trace element B solution is obtained.

[0101] Step 4: Prepare the culture medium solution:

[0102] Add 900 parts of lignocellulase hydrolyzate, 2 parts of corn steep liquor, 3 parts of K2HPO4, 3 parts of NaH2PO4, 1 part of MgSO4·7H2O, 1 part of ammonium sulfate, 1 part of trace element A solution, and 1 part of trace element B solution to the culture medium to obtain the culture medium solution.

[0103] Step 5: After activating Methylobacillus flagellatus ATCC 27329, inoculate it into the culture medium, and add Vitreoscilla hemoglobin and stir for high-density fermentation. After 60 h, a mixed solution A is obtained; the fermentation temperature is 30 °C; the rotation speed is 150 r / min;

[0104] After 5 h of fermentation, add a mixture C composed of phenoxyquinoline, methanol, formamide, ferrous sulfate, calcium chloride, and Triton X-100 to the culture medium. The mass ratio of the mixture C to the mixed solution A is 6:10;

[0105] For the mixture C, any common feeding method in the art can be used. Here, taking the common batch feeding method in the art as an example, after 4 h of fermentation, add 3% of the mass of the fermentation broth of the mixture C to the culture medium every 100 min. For every 100,000 parts of the fermentation broth, add 5 parts of phenoxyquinoline, 2000 parts of methanol, 200 parts of formamide, 70 parts of ferrous sulfate, 40 parts of calcium chloride, 2000 parts of Triton X-100, and 10,000 parts of water. Among them, phenoxyquinoline is used as a PQQ-dependent methanol dehydrogenase inhibitor, methanol is used as a carbon source, formamide is used as a nitrogen source, ferrous sulfate and calcium chloride are used as cofactors, and Triton X-100 is used as a cell membrane permeabilizer; the batch feeding is a common feeding method in the art to maintain the dynamic balance of nutrients in the fermentation broth, which can avoid substrate inhibition and thus significantly increase the yield;

[0106] Among them, the mass ratio of Vitreoscilla hemoglobin to the culture medium solution is 5:10000.

[0107] Step 6: Add 0.5 parts by mass of lysozyme to the mixed solution A to promote the release of intracellular pyrroloquinoline quinone, and obtain a fermentation broth containing PQQ.

[0108] Step 7: Measure the content of PQQ in the fermentation broth.

[0109] Example 5

[0110] A method for high-yield fermentation of pyrroloquinoline quinone using Methylobacillus flagellatus, the steps are as follows:

[0111] Step 1: Prepare the lignocellulose enzyme hydrolysate:

[0112] Add 100 g of lignocellulose to 1 L of water, and add a complex cellulase to obtain the lignocellulose enzyme hydrolysate;

[0113] Among them, the substrate dry weight of the cellulase is 45 FPU / g; the pH of the phosphate buffer solution used is 7.5, and the reaction temperature is 28 °C.

[0114] Step 2: Prepare the trace element A solution:

[0115] Add 1,000,000 parts of water, 10 parts of CaCl₂·2H₂O, 15.75 parts of ZnSO₄·7H₂O, 3.15 parts of MnCl₂, 0.525 parts of CuSO₄·5H₂O, and 1 part of NaCl to a container, and stir evenly to obtain the trace element A solution.

[0116] Step 3: Prepare the trace element B solution:

[0117] Add 1,000,000 parts of water, 10 parts of (NH₄)₆Mo₇O 24 ·4H₂O, 10 parts of KI, 10 parts of CoCl₂·6H₂O, and 10 parts of H₃BO₃ to a container, and stir evenly to obtain the trace element B solution.

[0118] Step 4: Prepare the culture medium solution:

[0119] Add 900 parts of the lignocellulose enzyme hydrolysate, 2 parts of corn steep liquor, 5 parts of K₂HPO₄, 5 parts of NaH₂PO₄, 1 part of MgSO₄·7H₂O, 5 parts of ammonium sulfate, 1 part of the trace element A solution, and 1 part of the trace element B solution to the culture medium to obtain the culture medium solution.

[0120] Step 5: After activating Methylobacterium extorquens DSM 1340, inoculate it into the culture medium and stir for high-density fermentation. After 70 h, obtain the mixed solution A; among them, the fermentation temperature is 30 °C; the rotation speed is 150 r / min;

[0121] After 5 h of fermentation, add a mixture C composed of quinoxyfen, methanol, formamide, ferrous sulfate, calcium chloride, and Triton X-100 to the culture medium. The mass ratio of the mixture C to the mixed solution A is 6.5:10;

[0122] Mixture C can be added by any common feeding method in the art. Here, taking the common batch feeding method in the art as an example, after 5 hours of fermentation, 2% of the mass of the fermentation broth of mixture C is fed into the culture medium every 110 minutes. For every 100,000 parts of the fermentation broth, 10 parts of phenoxyquinoline, 4000 parts of methanol, 400 parts of formamide, 60 parts of ferrous sulfate, 40 parts of calcium chloride, 2000 parts of Triton X-100, and 10,000 parts of water are added. Among them, phenoxyquinoline is used as a PQQ-dependent methanol dehydrogenase inhibitor, methanol is used as a carbon source, formamide is used as a nitrogen source, ferrous sulfate and calcium chloride are used as cofactors, and Triton X-100 is used as a cell membrane permeabilizer; the batch feeding is a common feeding method in the art to maintain the dynamic balance of nutrients in the fermentation broth, which can avoid substrate inhibition and thus significantly increase the yield;

[0123] Step Six: Add 0.7 parts by mass of lysozyme to the mixed solution A to promote the release of intracellular pyrroloquinoline quinone, and obtain a fermentation broth containing PQQ.

[0124] Step Seven: Measure the content of PQQ in the fermentation broth.

[0125] Example 6

[0126] A method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens, the steps are as follows:

[0127] Step One: Prepare a lignocellulose enzyme hydrolysate:

[0128] Add 100 g of lignocellulose to 1 L of water, and add a complex cellulase to obtain a lignocellulose enzyme hydrolysate;

[0129] Among them, the substrate dry weight of the cellulase is 45 FPU / g; the pH of the phosphate buffer solution used is 7.5, and the reaction temperature is 28 °C.

[0130] Step Two: Prepare a trace element A solution:

[0131] Add 1,000,000 parts of water, 10 parts of CaCl2·2H2O, 15.75 parts of ZnSO4·7H2O, 3.15 parts of MnCl2, 0.525 parts of CuSO4·5H2O, and 1 part of NaCl to a container, and stir evenly to obtain a trace element A solution.

[0132] Step Three: Prepare a trace element B solution:

[0133] Add 1,000,000 parts of water, 10 parts of (NH4)6Mo7O 24 ·4H2O, 10 parts of KI, 10 parts of CoCl2·6H2O, and 10 parts of H3BO3 to a container, and stir evenly to obtain a trace element B solution.

[0134] Step 4: Prepare the culture medium solution:

[0135] Add 900 parts of lignocellulase hydrolyzate, 2 parts of corn steep liquor, 4 parts of K2HPO4, 5 parts of NaH2PO4, 2 parts of MgSO4·7H2O, 7 parts of ammonium sulfate, 1 part of trace element A solution, and 1 part of trace element B solution to the culture medium to obtain the culture medium solution.

[0136] Step 5: After activating Methylobacterium extorquens DSM 1340, inoculate it into the culture medium, and add Vitreoscilla hemoglobin and stir for high-density fermentation. After 70 h, obtain mixture A; the fermentation temperature is 30 °C; the rotation speed is 150 r / min;

[0137] After 5 h of fermentation, add mixture C composed of phenoxyquinoline, methanol, formamide, ferrous sulfate, calcium chloride, and Triton X-100 to the culture medium. The mass ratio of mixture C to mixture A is 6.5:10;

[0138] Mixture C can be added by any common feeding method in the art. Here, taking the common batch feeding method in the art as an example, after 5 h of fermentation, add 4% of the mass of the fermentation broth of mixture C to the culture medium every 120 min. Add 10 parts of phenoxyquinoline, 4000 parts of methanol, 400 parts of formamide, 60 parts of ferrous sulfate, 40 parts of calcium chloride, 2000 parts of Triton X-100, and 10000 parts of water to every 100000 parts of the fermentation broth. Among them, phenoxyquinoline is used as a PQQ-dependent methanol dehydrogenase inhibitor, methanol is used as a carbon source, formamide is used as a nitrogen source, ferrous sulfate and calcium chloride are used as cofactors, and Triton X-100 is used as a cell membrane permeabilizer; the batch feeding is a common feeding method in the art to maintain the dynamic balance of nutrients in the fermentation broth, which can avoid substrate inhibition and thus significantly increase the yield;

[0139] Among them, the mass ratio of Vitreoscilla hemoglobin to the culture medium solution is 5:10000.

[0140] Step 6: Add 0.7 parts by mass of lysozyme to mixture A to promote the release of intracellular pyrroloquinoline quinone and obtain a fermentation broth containing PQQ.

[0141] Step 7: Measure the content of PQQ in the fermentation broth.

[0142] Example 7

[0143] A method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens, the steps are as follows:

[0144] Step 1: Prepare lignocellulase hydrolyzate:

[0145] Add 100 g of lignocellulose to 1 L of water, add complex cellulase, and obtain lignocellulase hydrolyzate;

[0146] Among them, the dry weight of the substrate of cellulase is 45 FPU / g; the pH of the used phosphate buffer solution is 7.5, and the reaction temperature is 28 °C.

[0147] Step 2: Prepare trace element A solution:

[0148] Add 1,000,000 parts of water, 10 parts of CaCl₂·2H₂O, 15.75 parts of ZnSO₄·7H₂O, 3.15 parts of MnCl₂, 0.525 parts of CuSO₄·5H₂O, and 1 part of NaCl to a container, and stir evenly to obtain trace element A solution.

[0149] Step 3: Prepare trace element B solution:

[0150] Add 1,000,000 parts of water, 10 parts of (NH₄)₆Mo₇O 24 ·4H₂O, 10 parts of KI, 10 parts of CoCl₂·6H₂O, and 10 parts of H₃BO₃ to a container, and stir evenly to obtain trace element B solution.

[0151] Step 4: Prepare the culture medium solution:

[0152] Add 900 parts of lignocellulose enzyme hydrolysate, 2 parts of corn steep liquor, 6 parts of K₂HPO₄, 5 parts of NaH₂PO₄, 1 part of MgSO₄·7H₂O, 2 parts of ammonium sulfate, 1 - 2 parts of trace element A solution, and 0.5 - 1 part of trace element B solution to the culture medium to obtain the culture medium solution.

[0153] Step 5: After activating Methylobacterium extorquens ATCC 43645, inoculate it into the culture medium, and add Vitreoscilla hemoglobin and stir for high - density fermentation. After 70 h, obtain mixture A; among them, the fermentation temperature is 30 °C; the rotation speed is 150 r / min;

[0154] After fermenting for 5 h, add mixture C composed of phenoxyquinoline, methanol, formamide, ferrous sulfate, calcium chloride, and Triton X - 100 to the culture medium. The mass ratio of mixture C to mixture A is 6.5:10;

[0155] Mixture C can be added by any common feeding method in the art. Here, taking the common batch feeding method in the art as an example, after 5 h of fermentation, 4% of the mass of the fermentation broth of mixture C is fed into the culture medium every 120 min. For every 100,000 parts of the fermentation broth, 10 parts of phenoxyquinoline, 4000 parts of methanol, 400 parts of formamide, 60 parts of ferrous sulfate, 40 parts of calcium chloride, 2000 parts of Triton X-100, and 10,000 parts of water are added. Among them, phenoxyquinoline is used as a PQQ-dependent methanol dehydrogenase inhibitor, methanol is used as a carbon source, formamide is used as a nitrogen source, ferrous sulfate and calcium chloride are used as cofactors, and Triton X-100 is used as a cell membrane permeabilizer; the batch feeding is a common feeding method in the art to maintain the dynamic balance of nutrients in the fermentation broth, which can avoid substrate inhibition and thus significantly increase the yield;

[0156] Among them, the mass ratio of Vitreoscilla hemoglobin to the culture medium solution is 5:10000.

[0157] Step six: Add 0.7 parts by mass of lysozyme to the mixed solution A to promote the release of intracellular pyrroloquinoline quinone, and obtain a fermentation broth containing PQQ.

[0158] Step seven: Determine the content of PQQ in the fermentation broth.

[0159] Example 8

[0160] A method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens, the steps are as follows:

[0161] Step one: Prepare a lignocellulase hydrolyzate:

[0162] Add 100 g of lignocellulose to 1 L of water, and add a composite cellulase to obtain a lignocellulase hydrolyzate;

[0163] Among them, the substrate dry weight of the cellulase is 45 FPU / g; the pH of the used phosphate buffer solution is 7.5, and the reaction temperature is 28 °C.

[0164] Step two: Prepare a trace element A solution:

[0165] Add 1,000,000 parts of water, 10 parts of CaCl2·2H2O, 15.75 parts of ZnSO4·7H2O, 3.15 parts of MnCl2, 0.525 parts of CuSO4·5H2O, and 1 part of NaCl to a container, and stir evenly to obtain a trace element A solution.

[0166] Step three: Prepare a trace element B solution:

[0167] Add 1,000,000 parts of water, 10 parts of (NH4)6Mo7O 24· 4H2O, 10 parts of KI, 10 parts of CoCl2·6H2O, 10 parts of H3BO3, and after stirring evenly, a trace element B solution is obtained.

[0168] Step 4: Prepare the culture medium solution:

[0169] Add 900 parts of lignocellulase hydrolysate, 2 parts of corn steep liquor, 5 parts of K2HPO4, 5 parts of NaH2PO4, 2 parts of MgSO4·7H2O, 5 parts of ammonium sulfate, 1 part of trace element A solution, and 1 part of trace element B solution to the culture medium to obtain the culture medium solution.

[0170] Step 5: After activating Methylobacillus flagellatus ATCC 43645, inoculate it into the culture medium, and add Vitreoscilla hemoglobin and stir for high-density fermentation. After 70 h, a mixed solution A is obtained; wherein the fermentation temperature is 30 °C; the rotation speed is 150 r / min;

[0171] After 5 h of fermentation, add a mixture C composed of phenoxyquinoline, methanol, formamide, ferrous sulfate, calcium chloride, and Triton X-100 to the culture medium. The mass ratio of the mixture C to the mixed solution A is 6.5:10;

[0172] For the mixture C, any common feeding method in the art can be selected. Here, taking the common batch feeding method in the art as an example, after 5 h of fermentation, add 5% of the mixture C of the fermentation broth mass to the culture medium every 110 min. For every 100,000 parts of the fermentation broth, add 10 parts of phenoxyquinoline, 4000 parts of methanol, 400 parts of formamide, 60 parts of ferrous sulfate, 40 parts of calcium chloride, 2000 parts of Triton X-100, and 10,000 parts of water. Among them, phenoxyquinoline is used as a PQQ-dependent methanol dehydrogenase inhibitor, methanol is used as a carbon source, formamide is used as a nitrogen source, ferrous sulfate and calcium chloride are used as cofactors, and Triton X-100 is used as a cell membrane permeabilizer; the batch feeding is a common feeding method in the art to maintain the dynamic balance of nutrients in the fermentation broth, which can avoid substrate inhibition and thus significantly increase the yield;

[0173] Among them, the mass ratio of Vitreoscilla hemoglobin to the culture medium solution is 5:10000.

[0174] Step 6: Add 0.7 mass parts of lysozyme to the mixed solution A to promote the release of intracellular pyrroloquinoline quinone, and obtain a fermentation broth containing PQQ.

[0175] Step 7: Measure the content of PQQ in the fermentation broth.

[0176] Comparative Example 1

[0177] A method for high-yield fermentation of pyrroloquinoline quinone using Methylobacillus flagellatus, the steps are as follows:

[0178] Step 1: Prepare the lignocellulose enzymatic hydrolysate:

[0179] Add 100 g of lignocellulose to 1 L of water, and add complex cellulase to obtain the lignocellulose enzymatic hydrolysate;

[0180] Among them, the substrate dry weight of the cellulase is 45 FPU / g; the pH of the used phosphate buffer solution is 7.5, and the reaction temperature is 28 °C.

[0181] Step 2: Prepare trace element A solution:

[0182] Add 1,000,000 parts of water, 10 parts of CaCl₂·2H₂O, 15 parts of ZnSO₄·7H₂O, 3 parts of MnCl₂, 0.5 part of CuSO₄·5H₂O, and 1 part of NaCl to the container, and stir evenly to obtain the trace element A solution.

[0183] Step 3: Prepare trace element B solution:

[0184] Add 1,000,000 parts of water, 10 parts of (NH₄)₆Mo₇O 24 ·4H₂O, 15 parts of KI, 15 parts of CoCl₂·6H₂O, and 10 parts of H₃BO₃ to the container, and stir evenly to obtain the trace element B solution.

[0185] Step 4: Prepare the culture medium solution:

[0186] Add 900 parts of lignocellulose enzymatic hydrolysate, 2 parts of corn steep liquor, 3 parts of K₂HPO₄, 3 parts of NaH₂PO₄, 1 part of MgSO₄·7H₂O, 1 part of ammonium sulfate, 1 part of trace element A solution, and 1 part of trace element B solution to the culture medium to obtain the culture medium solution.

[0187] Step 5: After activating Methylobacterium extorquens ATCC 27329, inoculate it into the culture medium, and add Vitreoscilla hemoglobin and stir for high-density fermentation. After 70 h, obtain the fermentation broth; among them, the fermentation temperature is 30 °C; the rotation speed is 150 r / min; the mass ratio of Vitreoscilla hemoglobin to the culture medium solution is 5:10,000.

[0188] Step 6: Add 0.5 part by mass of lysozyme to the mixed solution A to promote the release of intracellular pyrroloquinoline quinone, and obtain the fermentation broth containing PQQ.

[0189] Step 7: Measure the content of PQQ in the fermentation broth.

[0190] Comparative Example 2

[0191] A method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens, the steps are as follows:

[0192] Step 1: Prepare the lignocellulose enzymatic hydrolysate:

[0193] Add 100 g of lignocellulose to 1 L of water, and add complex cellulase to obtain the lignocellulose enzymatic hydrolysate;

[0194] Among them, the substrate dry weight of the cellulase is 45 FPU / g; the pH of the used phosphate buffer solution is 7.5, and the reaction temperature is 28 °C.

[0195] Step 2: Prepare trace element A solution:

[0196] Add 1,000,000 parts of water, 10 parts of CaCl₂·2H₂O, 15.75 parts of ZnSO₄·7H₂O, 3.15 parts of MnCl₂, 0.525 parts of CuSO₄·5H₂O, and 1 part of NaCl to a container, and stir evenly to obtain the trace element A solution.

[0197] Step 3: Prepare trace element B solution:

[0198] Add 1,000,000 parts of water, 10 parts of (NH₄)₆Mo₇O 24 ·4H₂O, 10 parts of KI, 10 parts of CoCl₂·6H₂O, and 10 parts of H₃BO₃ to a container, and stir evenly to obtain the trace element B solution.

[0199] Step 4: Prepare the culture medium solution:

[0200] Add 900 parts of lignocellulose enzymatic hydrolysate, 2 parts of corn steep liquor, 3 parts of K₂HPO₄, 3 parts of NaH₂PO₄, 3 parts of MgSO₄·7H₂O, 8 parts of ammonium sulfate, 1 part of trace element A solution, and 1 part of trace element B solution to the culture medium to obtain the culture medium solution.

[0201] Step 5: After activating Methylobacterium extorquens ATCC 27329, inoculate it into the culture medium, and add Vitreoscilla hemoglobin and stir for high-density fermentation. After 70 h, obtain the mixed solution A; among them, the fermentation temperature is 30 °C; the rotation speed is 150 r / min; the mass ratio of Vitreoscilla hemoglobin to the culture medium solution is 5:10000.

[0202] Step 6: Add 0.7 mass parts of lysozyme to the mixed solution A to promote the release of intracellular pyrroloquinoline quinone, and obtain the fermentation broth containing PQQ.

[0203] Step 7: Determine the content of PQQ in the fermentation broth.

[0204] Example of implementation effect 1

[0205] Detect PQQ in the fermentation broth by HPLC method

[0206] HPLC detection method: In the mobile phase, the volume ratio of methanol: water: trifluoroacetic acid is 60:40:0.1; the injection volume is 1 μL; the flow rate is 0.5 mL / min; the detection wavelength is 249 nm; the column temperature is 30 °C; the detector is DAD; the chromatographic column is C 18 AQ column.

[0207] Prepare a 1 mg / mL PQQ standard solution, and gradient dilute the standard solution to standard solutions of 50, 100, 200, 300, and 400 mg / L. Determine the standard curve with the 5 groups of diluted standard solutions. The standard curve is as Figure 2 shown. Use the HPLC detection method to perform high-performance liquid chromatography detection on the above standard solutions. The obtained data uses the peak area of the chromatogram as the ordinate (Y) and the mass concentration as the abscissa (X) to obtain the peak area-concentration standard curve. Thus, the yield of PQQ in the fermentation broth is calculated as shown in Table 1.

[0208] Table 1 PQQ content in the fermentation broth of each example

[0209]

[0210] Detect the PQQ content in the fermentation broth of each example. The results are shown in Table 1. By fermenting and culturing Methylobacterium extorquens with a specific lignocellulose hydrolate medium, adding phenoxyquinoline with an inhibitory effect on PQQ-dependent methanol dehydrogenase, carbon source methanol, nitrogen source formamide, cofactors ferrous sulfate and calcium chloride, and cell membrane permeabilizer Triton X-100 to the medium, the yield of pyrroloquinoline quinone can be increased by 5-10 times; in addition, the preparation method of the present invention is simple and low-cost, and is suitable for large-scale industrial production.

[0211] Example of implementation effect 2

[0212] Separation and purification of pyrroloquinoline quinone:

[0213] By comparing with the standard product, the PQQ standard product chromatogram is as Figure 4 shown, and the HPLC chromatogram of PQQ in the fermentation broth is as Figure 3 shown. The peak emergence times are the same, indicating that the fermentation broth contains PQQ. Concentrate the obtained fermentation supernatant to one-fourth of the original volume, then filter and first pass through an HP-20 column, and then elute with 3 column volumes of deionized water, 5% ethanol solution, 20% ethanol solution, and pure ethanol in sequence to obtain a preliminarily purified PQQ product that is red. Then pass the obtained preliminarily purified PQQ product through an ODS column and elute it in the same way, and collect the eluted solutions separately to obtain a re-purified PQQ product.

[0214] Identification of the purified product

[0215] The obtained product was analyzed by mass spectrometry. Analysis conditions: Ion source: ESI, Desolvation temperature scanning mode: Anion, Dry gas flow rate: 5 L / min, Sheath gas temperature: 350 °C, Capillary voltage: 2500 V, Sheath gas flow rate: 500 L / h, Collision energy: 10 V, Cone voltage: 2.5 V, Mass scanning range: 700 m / z, Acquisition once every 0.2 s.

[0216] The purified product at a concentration of 1 mg / L was scanned using a UV-Vis spectrophotometer, with a scanning range of 190 - 365 nm.

[0217] Product analysis: The purified product was identified by mass spectrometry and UV spectroscopy. The results are as Figure 5 shown. The molecular ion peak [M+NH4] + in the mass spectrum is 348.11, determining the molecular weight to be 330, which is consistent with the molecular weight of PQQ reported in the literature. Figure 6 The UV spectrum in the literature shows that the UV absorption peaks of the purified product are 248 and 330 nm, which is Figure 1 consistent with the UV spectrum of PQQ reported in the literature.

[0218] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens, characterized in that: Methylobacterium extorquens was inoculated into a culture medium, and then Vitreoscilla hemoglobin was added and stirred for fermentation to obtain a mixed solution A. Lysozyme was added to the mixed solution A to obtain a fermentation solution rich in PQQ.

2. The method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens according to claim 1, characterized in that: The mass ratio of the Vitreoscilla hemoglobin to the culture medium is 4-10:10000; and the amount of lysozyme added to the mixed solution A is 0.2-0.7 parts by mass.

3. The method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens according to claim 2, characterized in that: The culture medium components include 900-1000 parts of lignocellulose enzymatic hydrolysate, 2-5 parts of corn steep liquor, 2-6 parts of K2HPO4, 3-8 parts of NaH2PO4, 0.05-3 parts of MgSO4·7H2O, 1-9 parts of ammonium sulfate, 1-2 parts of trace element A solution and 0.5-1 part of trace element B solution by mass.

4. The method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens according to claim 3, characterized in that: The trace element A solution comprises, by mass, 1,000,000-1,500,000 parts of water, 5-20 parts of CaCl2.2H2O, 10-20 parts of ZnSO4.7H2O, 2-4 parts of MnCl2, 0.1-1 parts of CuSO4.5H2O and 1-3 parts of NaCl.

5. The method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens according to claim 4, characterized in that: The trace element B solution comprises 1,000,000-1,500,000 parts of water, 10-15 parts of (NH4)6Mo7O 24 ·4H2O, 10-15 parts of KI, 10-15 parts of CoCl2·6H2O and 10-15 parts of H3BO3.

6. The method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens according to claim 5, characterized in that: The specific steps are as follows: Step 1: Preparation of enzymatic hydrolysate of lignocellulose: Add 80-150 g of lignocellulose to 1 L of water, add complex cellulase to react, and obtain lignocellulose enzymatic hydrolyzate; Step 2: Prepare trace element A solution: Add 1,000,000-1,500,000 parts of water, 5-20 parts of CaCl2·2H2O, 10-20 parts of ZnSO4·7H2O, 2-4 parts of MnCl2, 0.1-1 parts of CuSO4·5H2O and 1-3 parts of NaCl into a container, stir evenly to obtain a trace element A solution; Step 3: Prepare trace element B solution: Add 1,000,000-1,500,000 parts of water, 10-15 parts of (NH4)6Mo7O 24 ·4H2O, 10-15 parts of KI, 10-15 parts of CoCl2·6H2O and 10-15 parts of H3BO3, stir evenly to obtain a trace element B solution; Step 4: Prepare the culture medium solution: Adding 900-1000 parts of lignocellulose enzymatic hydrolyzate, 2-5 parts of corn steep liquor, 2-6 parts of K2HPO4, 3-8 parts of NaH2PO4, 0.05-3 parts of MgSO4·7H2O, 6-9 parts of ammonium sulfate, 1-2 parts of trace element A solution, and 0.5-1 parts of trace element B solution into a culture medium to obtain a culture medium solution; Step 5: activating Methylobacterium extorquens, inoculating it into the culture medium, adding Vitreoscilla hemoglobin, stirring and fermenting to obtain a mixed solution A; Step 6: Add lysozyme to the mixed solution A to obtain a fermentation solution containing PQQ.

7. The method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens according to claim 6, characterized in that: The activity of the cellulase in the enzymatic hydrolysate of the lignocellulose is 40-50 FPU / g.

8. The method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens according to claim 7, characterized in that: The method also includes adding a mixture C consisting of phenoxyquinoline, methanol, formamide, ferrous sulfate, calcium chloride and Triton X-100 to the culture medium after stirring and fermenting for 4-6 hours; the mass ratio of the mixture C to the mixed solution A is 6-7:

10.

9. The method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens according to claim 8, characterized in that: The stirring fermentation time is 60-80 hours, and the temperature is 20-35°C.

10. Use of the method for high-yield fermentation of pyrroloquinoline quinone using Methylobacterium extorquens according to any one of claims 1 to 9 in the industrial production of pyrroloquinoline quinone.

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