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CN113337432B - Methylophilus for producing pyrroloquinoline quinone and application thereof - Google Patents

Methylophilus for producing pyrroloquinoline quinone and application thereof Download PDF

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CN113337432B
CN113337432B CN202110626696.9A CN202110626696A CN113337432B CN 113337432 B CN113337432 B CN 113337432B CN 202110626696 A CN202110626696 A CN 202110626696A CN 113337432 B CN113337432 B CN 113337432B
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pyrroloquinoline quinone
fermentation
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pqq
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乔莉苹
周宁
李帆
王秀娟
沈延臻
郭学平
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Bloomage Biotech Co Ltd
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Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a methyl bacterium (methyl bacterium sp) HQ-1 with a preservation number of CCTCCNO: m2021252. The invention also relates to an application of the methylovorus (sp) HQ-1 in fermenting and producing pyrroloquinoline quinone, and a method and a composition for preparing pyrroloquinoline quinone. The strain of methylotrophic bacteria (methyl sp) HQ-1 provided by the invention is aerobically cultured for 1-2 days in a culture medium which takes methanol as a carbon source and peptone as a nitrogen source, and the yield of pyrroloquinoline quinone can reach 100mg/L at the shake flask level. The strain screened by the invention can secrete PQQ to the outside of cells, has the advantage of rapid growth compared with common PQQ producing strains, and provides a new strain for industrialized fermentation production of PQQ.

Description

Methylophilus for producing pyrroloquinoline quinone and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a methyl bacterium for producing pyrroloquinoline quinone and application thereof.
Background
Pyrroloquinoline quinone (Pyrroloquinoline quinone, PQQ, also known as Methoxatin) is a water-soluble quinone compound, has thermostability, is first identified in bacteria as a cofactor for alcohol and glucose dehydrogenase, is the third prosthetic group found in bacterial dehydrogenase following flavin nucleotides and nicotinamide nucleotides, and is known in the world medical community as the fourteenth vitamin. PQQ is a novel water-soluble vitamin, is an oxidation-reduction enzyme prosthetic group, is very rare, exists in the tissues of some microorganisms, plants and animals, and has the phenomenon of low reproductive capacity of experimental mice lacking the substance. Studies have shown that PQQ is found in everyday vegetables, fruits such as natto, celery, kiwi, etc. The substances are present in viscera and body fluid of human body, and the content of the substances is 0.8-5.9ng/g, wherein the content of spleen is the highest and is 5.9ng/g. The content of PQQ and its derivatives in human milk is up to 140-180mg/L, which is tens times higher than that of ordinary food, and it is proved that the PQQ and its derivatives can play a vital role in the growth of newborn infants, and in addition, the PQQ content in human milk is 50 times that of animal and plant milk such as milk. It is generally believed that PQQ can only be synthesized in certain gram-negative bacteria, and that PQQ cannot be synthesized in plants and animals, and is mainly obtained by the dietary route.
The research shows that the PQQ has the functions of resisting oxidation, whitening, promoting amino acid absorption, preventing and treating senile dementia, conditioning nerve diseases, diminishing inflammation, protecting heart, preventing and treating cataract, resisting cancer, treating liver diseases and the like, and has good development prospect in the fields of health care products, cosmetics, medicines and the like.
Currently, there are two methods for producing PQQ, namely chemical synthesis and microbial fermentation. The production of the PQQ is realized firstly by a chemical synthesis method, and at present, the chemical synthesis of the PQQ needs 16 steps of chemical synthesis reaction including 11 steps of chemical synthesis reaction and 5 steps of separation and purification, so that the PQQ with the purity of about 97% can be obtained, the chemical synthesis process is complex, the byproducts are more, the purification technology is difficult, and the synthesis cost is high. Compared with the chemical synthesis method of PQQ, the microbial fermentation method gradually becomes a new research direction, has the advantages of environmental protection, low cost, few steps, easy separation and the like, but also has the problems of lack of excellent industrial production strains, immature fermentation strategies and the like.
At present, PQQ is reported to be found in various strains, the highest yield of the PQQ obtained by fermentation reported in the prior art can reach 2g/L, but most of reported yields are not high, so that the screening of high-yield strains is still the key of industrial production of the PQQ.
Patent CN102061278B discloses the application of methylotrophic bacteria (methyl sp.) MP688 to produce pyrroloquinoline quinone (PQQ), the PQQ yield reaches 125mg/L, but the strain has a cultivation time of 5 days, is relatively long, and has a certain difficulty in realizing industrial scale production.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a strain of methyl bacterium (methyl vorus. Sp) HQ-1 which is preserved in China Center for Type Culture Collection (CCTCC) for China, wherein the preservation address is China university of Wuhan, and the preservation number is CCTCC NO: m2021252. The invention also provides a composition of the methylotrophic bacteria and the fermentation broth, and a method for producing the PQQ by fermentation.
The invention separates and screens a strain of methyl bacterium (methyl bacterium sp) HQ-1 from chemical sewage, the fermentation period is about 1-2 days, the invention has the advantage of rapid growth, and the yield is about 100mg/L through preliminary optimization of carbon and nitrogen sources and HPLC verification.
The technical scheme of the invention is as follows:
1. methylovorus (sp) HQ-1, accession number: cctccc NO: m2021252.
2. The M.methylotrophicus HQ-1 according to item 1, wherein the 16s rRNA gene sequence is shown in SEQ ID NO:1
(GCTCAGATTGAACGCTGGCGGCATGCTTTACACATGCAAGTCGAACGGTAACAG AGAGAAGCTTGCTTCTCTGCTGACGAGTGGCGAACGGGTGAGTAATATATCGGAACGTACCGTATTGTGGGGGATAACTAGTCGAAAGATTAGCTAATACCGCATACGCCCTGAGGGGGAAAGTAGGGGATCTTCGGACCTTACGCAGAACGAGCGGCCGATATCTGATTAGCTAGTTGGTGGGGTAAAGGCCCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTCGCAAGGAAAGAAAACTTACTCGCTAATACCGGGTGAGGATGACGGTACCTTGATAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGAGCGCAGGCGGTTTTGTAAGTCAGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCGTTTGAAACTGCAAGGCTAGAGTATGGGAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCAATGGCGAAGGCAGCCCCCTGGCCTAATACTGACGCTCATGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCTACTAGTTGTTGGTGGAGTAAAATCCATTAGTAACGCAGCTAACGCGTGAAGTAGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGATTATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTGGCCTTGACATGCTACTAACGAAGCAGAGATGCATTAGGTGCCCGAAAGGGAAAGTAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGCCAATAATTGCCATCATTTAGTTGGGCACTTTATTGGGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGCCAGGGCTTCACACGTAATACAATGGTCGGTACAGAGGGTTGCCAACCCGCGAGGGGGAGCCAATCCCAGAAAGCCGATCGTAGTCCGGATTGCAGTCTGCAACTCGACTGCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTCTACCAGAAGTAGTTAGTCTAACCGCAAGGGGGACGATTACCACGGTAGGATTCATGACTGGGGTGAAGTCGT)。
3. The application of the methylotrophic bacterium HQ-1 in the item 1 or 2 in the production of pyrroloquinoline quinone.
4. A composition comprises Methylophilus HQ-1 and a fermentation broth, wherein the concentration of pyrroloquinoline quinone in the fermentation broth is more than 10.00mg/L, specifically more than 20.00mg/L, more than 30.00mg/L, more than 40.00mg/L, more than 50.00mg/L, more than 60.00mg/L, more than 70.00mg/L, more than 80.00mg/L, more than 90.00mg/L, more than 100.00mg/L, and preferably more than 40.00 mg/L.
5. A method of producing pyrroloquinoline quinone, comprising: a pyrroloquinoline quinone produced by fermentation using the M.methylotrophicus HQ-1 described in item 1 or 2.
6. The method according to item 5, comprising:
activating the methyl-eating bacteria HQ-1 and inoculating the activated methyl-eating bacteria HQ-1 into a seed culture medium;
then inoculating the obtained seed liquid into a fermentation medium according to the volume ratio of 1-10% for fermentation culture to obtain pyrroloquinoline quinone.
7. The method according to item 6, comprising:
the activation is to culture in a slant culture medium for 12-24 hours at 25-32 ℃; and/or the number of the groups of groups,
the seed liquid is obtained by shaking culture in a seed culture medium at 25-32 ℃ and 120-220 rpm for 12-24 hours; and/or the number of the groups of groups,
the fermentation culture is carried out in a fermentation culture medium for 24-48 hours under the conditions of 25-32 ℃ and the rotating speed of 120-220 rpm.
8. The method of item 6, wherein each 1L of the seed medium comprises: 3-10 g of ammonium sulfate, 5-20 g of methanol and KH 2 PO 4 1.5～3g、Na 2 HPO 4 ·12H 2 O 5～10g、MgSO 4 ·7H 2 0.1 to 0.5g of O, 0.01 to 0.05g of ferric citrate and CaCl 2 0.01～0.05g、MnCl 2 ·4H 2 O 0.1～0.5mg、ZnSO 4 ·7H 2 O 0.01～0.05g、CuSO 4 ·7H 2 0.1 to 0.5mg of O, 6.8 to 7.2 of pH and the balance of water.
9. The method of item 6, wherein each 1L of the fermentation medium comprises:
3-20 g of nitrogen source, 5-20 g of methanol, 0.5-2 g of tyrosine, 0.5-2 g of glutamic acid and KH 2 PO 4 1.5～3g、Na 2 HPO 4 ·12H 2 O 5～10g、MgSO 4 ·7H 2 0.1 to 0.5g of O, 0.01 to 0.05g of ferric citrate and CaCl 2 0.01～0.05g、MnCl 2 ·4H 2 O 0.1～0.5mg、ZnSO 4 ·7H 2 O 0.01～0.05g、CuSO 4 ·7H 2 0.1 to 0.5mg of O, 6.8 to 7.2 of pH and the balance of water.
Compared with the common PQQ producing bacteria, the methyl bacterium (methyl vorus. Sp) HQ-1 provided by the invention has the advantage of rapid growth, and the yield can reach 100mg/L on the shake flask level through preliminary optimization of carbon source and nitrogen source and HPLC verification.
Preservation of biological materials
Preservation date: 2021, 03, 19
Preservation unit: china Center for Type Culture Collection (CCTCC)
Preservation number: cctccc NO: m2021252
Drawings
Fig. 1: a photo of the morphological characteristics of Methylovorus (sp) HQ-1;
fig. 2: colony morphological feature photographs of methylotrophic bacteria (methyl vorus. Sp.) HQ-1;
fig. 3: HQ-1 culture and PQQ standard HPLC results;
fig. 4: results of 16s rRNA gene sequencing of Methylovorus (sp) HQ-1.
Detailed Description
The following embodiments of the invention are merely illustrative of specific embodiments for carrying out the invention and are not to be construed as limiting the invention. Any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the invention are intended to be equivalent arrangements which are within the scope of the invention.
As a specific embodiment of the invention, the invention relates to a methyl bacterium (methyl vorus sp) HQ-1 which is preserved in China Center for Type Culture Collection (CCTCC) at the year of 2021, 03 and 19, wherein the preservation address is the university of Wuhan, mail code: 430072 with the preservation number of CCTCC NO: m2021252, which can be used for the fermentative production of pyrroloquinoline quinone.
Pyrroloquinoline quinone, english name is methoxatin, PQQ for short, and molecular formula is C 14 H 6 N 2 O 8 Molecular weight 330.206, CAS registry number 72909-34-3, chemical intermediate. PQQ is a novel redox prosthetic group and has various physiological functions of resisting oxidation, regulating the level of free radicals in a body, promoting the regeneration of nerve growth factors and the like.
The 16s rRNA gene sequence of the methylotrophic bacterium HQ-1 is SEQ ID NO. 1, see figure 4.
rRNA has unique and important characteristics, and is structurally specific and conserved, so that it is resistant to mutations affecting its structure. The 16s rRNA gene has an inter-species polymorphic region, so that the sequence can be analyzed to determine the evolutionary distance and interrelation of various bacteria, identify the bacteria, and are ubiquitous in bacteria, so that the method is suitable for analysis of all bacteria.
The invention separates and screens a strain of methyl bacterium (methyl bacterium sp) HQ-1 from chemical sewage, has the fermentation period of about 1-2 days, has the advantage of quick growth, and can reach 100mg/L in shake flask level through preliminary optimization of carbon and nitrogen sources and HPLC verification.
As a specific embodiment of the present invention, the Methylophilus HQ-1 of the present invention is used for fermentative production of pyrroloquinoline quinone.
In a specific embodiment of the present invention, the present invention relates to a composition obtained by fermentation using the methylotrophic bacterium HQ-1 of the present invention, which comprises methylotrophic bacterium HQ-1 and a fermentation broth in which the pyrroloquinoline quinone concentration is 10.00mg/L or more, specifically 15.00mg/L, 20.00mg/L, 25.00mg/L, 30.00mg/L, 35.00mg/L, 40.00mg/L, 45.00mg/L, 50.00mg/L, 55.00mg/L, 60.00mg/L, 65.00mg/L, 70.00mg/L, 75.00mg/L, 80.00mg/L, 85.00mg/L, 90.00mg/L or 95.00mg/L, preferably 40.00mg/L or more.
In one embodiment, the invention relates to a method for producing pyrroloquinoline quinone, comprising: the pyrroloquinoline quinone is produced by fermentation using the aforementioned Methylophilus HQ-1.
In a specific embodiment, the methylotrophic bacterium HQ-1 is inoculated into a seed culture medium after activation; and then inoculating the obtained seed liquid into a fermentation culture medium according to the volume ratio of 1% -10% (which can be specifically 2%, 3%, 4%, 5%, 6%, 7%, 8% or 9%) for fermentation culture to obtain pyrroloquinoline quinone.
In a specific embodiment, the activation is performed in a slant culture medium under conditions of 25 to 32 ℃ (specifically, 26 ℃, 27 ℃, 28 ℃, 29 ℃,30 ℃ or 31 ℃) for 12 to 24 hours (specifically, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours or 23 hours).
The slant culture medium comprises the following components: 3-10 g of ammonium sulfate, 5-20 g of methanol and KH 2 PO 4 1.5～3g、Na 2 HPO 4 ·12H2O 5～10g、MgSO 4 ·7H 2 0.1 to 0.5g of O, 0.01 to 0.05g of ferric citrate and CaCl 2 0.01～0.05g、MnCl 2 ·4H 2 O 0.1～0.5mg、ZnSO 4 ·7H 2 O 0.01～0.05g、CuSO 4 ·7H 2 0.1 to 0.5mg of O, 6.8 to 7.2 of pH and the balance of water.
In a specific embodiment, the seed solution is obtained by shaking culture in a seed medium at 25 to 32℃which may be 26℃at 28℃at 29℃at 30℃at 31℃at 120 to 220rpm (which may be 130rpm, 140rpm, 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm or 210 rpm) for 12 to 24 hours (which may be 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours or 23 hours).
In a specific embodiment, the fermentation culture is carried out in a fermentation medium at a temperature of 25 to 32 ℃ (specifically, 26 ℃, 27 ℃, 28 ℃, 29 ℃,30 ℃ or 31 ℃) and a rotation speed of 120 to 220rpm (specifically, 130rpm, 140rpm, 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm or 210 rpm) for 24 to 48 hours (specifically, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours or 43 hours).
The methylotrophic bacteria (methyl esters. Sp.) HQ-1 provided in this example produced PQQ by fermentation with a growth cycle of about 1 to 2 days, and produced PQQ more rapidly than the conventional PQQ-producing bacteria.
In a specific embodiment, each 1L of the seed medium comprises: 3-10 g of ammonium sulfate, 5-20 g of methanol and KH 2 PO 4 1.5～3g、Na 2 HPO 4 ·12H2O 5～10g、MgSO 4 ·7H 2 0.1 to 0.5g of O, 0.01 to 0.05g of ferric citrate and CaCl 2 0.01～0.05g、MnCl 2 ·4H 2 O 0.1～0.5mg、ZnSO 4 ·7H 2 O 0.01～0.05g、CuSO 4 ·7H 2 0.1 to 0.5mg of O, 6.8 to 7.2 of pH and the balance of water.
The seed culture medium is used for spore germination, growth and mass propagation of mycelium, and makes the mycelium grow to be thick and strong, so that the seed with strong activity is obtained.
In one embodiment, each 1L of the fermentation medium comprises: 3-20 g of nitrogen source, 5-20 g of methanol, 0.5-2 g of tyrosine, 0.5-2 g of glutamic acid and KH 2 PO 4 1.5～3g、Na 2 HPO 4 ·12H 2 O 5～10g、MgSO 4 ·7H 2 0.1 to 0.5g of O, 0.01 to 0.05g of ferric citrate and CaCl 2 0.01～0.05g、MnCl 2 ·4H 2 O 0.1～0.5mg、ZnSO 4 ·7H 2 O 0.01～0.05g、CuSO 4 ·7H 2 0.1 to 0.5mg of O, 6.8 to 7.2 of pH and the balance of water.
The fermentation medium is used for the growth, propagation and synthesis of products. It can make seed inoculated and quickly grow to obtain a certain thallus concentration, and can make the grown thallus quickly synthesize the required product.
Carbon sources are a generic term for a type of nutrient that contains elemental carbon and is available for microbial growth and reproduction. The carbon source has the main effects of providing a carbon skeleton for cells, providing energy required for vital activities of the cells and providing a carbon skeleton for synthesis products. The carbon source plays an important role in preparing a bacterial culture medium and provides a material basis for normal growth and division of bacteria. Wherein the carbon source may be methanol.
Nitrogen sources are required for bacterial growth and product synthesis. The nitrogen source is mainly used for the synthesis of cell substances (amino acids, proteins, nucleic acids, etc.) and nitrogen-containing metabolites of bacteria. The nitrogen source used in the fermentation medium herein includes organic nitrogen sources and/or inorganic nitrogen sources. Wherein the nitrogen source can be one or more selected from peptone, yeast powder, soybean peptide, ammonium sulfate, beef extract, and corn flour.
Compared with the common PQQ producing bacteria, the methylotrophic bacteria (methyl vorus. Sp) HQ-1 provided by the application has the advantage of rapid growth, and can utilize complex nitrogen sources such as peptone, and the like, and the yield is about 100mg/L through preliminary optimization of carbon sources and nitrogen sources and HPLC verification.
Examples
The experimental methods used below are conventional methods if no special requirements are imposed.
Materials, reagents and the like used in the following are commercially available unless otherwise specified.
Screening the culture medium: each 1L of screening culture medium contains 3g of ammonium sulfate, 10g of methanol and KH 2 PO 4 1.5g、Na 2 HPO 4 ·12H 2 O 7.67g、MgSO 4 ·7H 2 O0.2g, ferric citrate 0.02g and CaCl 2 0.03 g、MnCl 2 ·4H 2 O 0.2mg、ZnSO 4 ·7H 2 O 0.02g、CuSO 4 ·7H 2 O0.2 mg, pH 7.0, balance water.
Seed culture medium: consistent with the composition of the screening medium.
Fermentation medium: each 1L of the fermentation medium contains 3g of nitrogen source, 10g of carbon source and casein
1.5g of amino acid, 1.5g of glutamic acid and KH 2 PO 4 1.4g、Na 2 HPO 4 ·12H 2 O 7.67g、MgSO 4 ·7H 2 0.2g of O, 0.02g of ferric citrate and CaCl 2 0.03 g、MnCl 2 ·4H 2 O 0.2mg、ZnSO 4 ·7H 2 O 0.02g、CuSO 4 ·7H 2 O0.2 mg, pH 7.0, balance water.
EXAMPLE 1 screening of PQQ-producing bacteria
Collecting 61 soil samples or water samples from Shandong, henan and inner Mongolia areas, enriching by a screening culture medium, diluting and coating on a screening culture medium plate, and culturing for 3-5 days at 30 ℃ to obtain a primary screening strain. The primary screening strains are respectively inoculated into test tubes filled with 5mL of screening culture medium, shake-cultured for 2 days at 30 ℃, then transferred into a triangular flask filled with 50mL of screening culture medium, and cultured for 3-5 days at 200rpm at 30 ℃. As shown in Table 1, the PQQ content of the fermentation supernatant was precisely determined by HPLC, wherein the strain No. Methylovorus sp HQ-1 was the highest in yield, up to 35.19mg/L.
TABLE 1 PQQ-producing different isolates
Strain numbering PQQ yield (mg/L) Strain numbering PQQ yield (mg/L)
HQ-1 35.19 HQ-8 18.32
HQ-2 22.32 HQ-9 5.43
HQ-3 15.18 HQ-10 10.45
HQ-4 12.03 HQ-11 20.14
HQ-5 3.26 HQ-12 13.45
HQ-6 2.87 HQ-13 11.08
HQ-7 17.65 HQ-14 1.35
Example 2 morphological observations
The bacterial form characteristics of Methylovorus (sp) HQ-1 are shown in figure 1. Specifically, the cells were observed to be in the form of short rods by observation with an OLYMPUS CX33 microscope.
The colony morphology characteristics of Methylovorus (sp) HQ-1 are shown in FIG. 2. Colonies were observed to be large, flat, moist, round, yellowish.
Example 3 identification of strains
The genome of the strain is sequenced and identified by a biological engineering (Shanghai) stock Co.Ltd, and the determination result of the 16s rRNA gene sequence of the methylotrophic bacterium HQ-1 is shown as SEQ ID NO. 1 (see figure 4 for details).
EXAMPLE 4 detection of product PQQ
The PQQ assay was performed by high performance liquid chromatography using the methyl vorus sp HQ-1 fermentation broth and the PQQ standard as test samples, with HPLC conditions of: sample injection amount 10 μl, column temperature 40 ℃, mobile phase (0.1% trifluoroacetic acid: acetonitrile=85:15), flow rate 0.6mL/min, detection wavelength 252nm, chromatographic column: eclips plus C18, by HPLC comparison, the HQ-1 broth and the PQQ standard gave complete agreement, as shown in FIG. 3.
Example 5 carbon optimization of fermentation Medium
Strain activation: the original strain was inoculated into a slant medium (identical to the seed medium described above) and cultured at 30℃for 20 hours.
Fermentation culture: the activated Methylovorus sp HQ-1 strain was inoculated into a seed medium, cultured with shaking at 30℃and 200rpm for 20 hours, and then the seed solution was inoculated into a flask containing a fermentation medium (the nitrogen source was ammonium sulfate) at 5% (v/v), 50mL of a 250mL flask solution, and fermented at 30℃and 200rpm for 2 days, wherein glucose, methanol, sucrose, fructose, lactose, glycerol and maltose were used as carbon sources of the fermentation medium, respectively. After completion, biomass (OD) 600 ) The PQQ content of the fermentation supernatant was rapidly determined by NBT-Gly chemistry, and the results are shown in Table 2.
TABLE 2 carbon source optimization results
Carbon source PQQ yield (mg/L) OD 600
Methanol 32.35 3.33
Glucose 0 0.27
Sucrose 0 0.19
Fructose 0 3.08
Lactose and lactose 0 0.15
Glycerol 0 0.16
Maltose 0 0.26
Example 6 Nitrogen Source optimization of fermentation Medium
Strain activation: the original strain was inoculated into a slant medium (identical to the seed medium described above) and cultured at 30℃for 20 hours.
Fermentation culture: the activated Methylovorus sp HQ-1 strain was inoculated to a seed medium, cultured with shaking at 32℃and 200rpm for 24 hours, and then the seed solution was inoculated to a flask containing a fermentation medium (methanol as a carbon source) at 3% (v/v), 50mL of a 250mL flask containing peptone, yeast powder, soybean peptide, ammonium sulfate, beef extract or corn powder as nitrogen sources of the fermentation medium, respectively, and fermented at 32℃and 200rpm for 2 days. After completion, biomass (OD) 600 ) The PQQ content of the fermentation supernatant was also rapidly determined by NBT-Gly chemistry, and the results are shown in Table 3.
TABLE 3 Nitrogen Source optimization results
Nitrogen source PQQ yield (mg/L) OD 600
Peptone 100 7.06
Yeast powder 60.15 4.12
Soybean peptide 56.12 3.18
Ammonium sulfate 22.82 3.15
Beef extract 40.18 4.00
Corn flour 23.15 2.98
Although embodiments of the present application have been described above, the present application is not limited to the specific embodiments and fields of application described above, which are merely illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous forms of the invention without departing from the scope of the invention as claimed.
Sequence listing
<110> Hua Xi Biotech Co., ltd
<120> a pyrroloquinoline quinone-producing methylotrophic bacterium and application thereof
<130> TPE01431
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1480
<212> DNA
<213> artificial sequence
<220>
<223> artificially synthesized
<400> 1
gctcagattg aacgctggcg gcatgcttta cacatgcaag tcgaacggta acagagagaa 60
gcttgcttct ctgctgacga gtggcgaacg ggtgagtaat atatcggaac gtaccgtatt 120
gtgggggata actagtcgaa agattagcta ataccgcata cgccctgagg gggaaagtag 180
gggatcttcg gaccttacgc agaacgagcg gccgatatct gattagctag ttggtggggt 240
aaaggcccac caaggcgacg atcagtagct ggtctgagag gacgaccagc cacactggaa 300
ctgagacacg gtccagactc ctacgggagg cagcagtggg gaattttgga caatgggcga 360
aagcctgatc cagccatgcc gcgtgagtga agaaggcctt cgggttgtaa agctctttcg 420
caaggaaaga aaacttactc gctaataccg ggtgaggatg acggtacctt gataagaagc 480
accggctaac tacgtgccag cagccgcggt aatacgtagg gtgcgagcgt taatcggaat 540
tactgggcgt aaagcgagcg caggcggttt tgtaagtcag atgtgaaagc cccgggctca 600
acctgggaac tgcgtttgaa actgcaaggc tagagtatgg gagagggggg tagaattcca 660
cgtgtagcag tgaaatgcgt agagatgtgg aggaatacca atggcgaagg cagccccctg 720
gcctaatact gacgctcatg ctcgaaagcg tggggagcaa acaggattag ataccctggt 780
agtccacgcc ctaaacgatg tctactagtt gttggtggag taaaatccat tagtaacgca 840
gctaacgcgt gaagtagacc gcctggggag tacggtcgca agattaaaac tcaaaggaat 900
tgacgggggc ccgcacaagc ggtggattat gtggattaat tcgatgcaac gcgaaaaacc 960
ttacctggcc ttgacatgct actaacgaag cagagatgca ttaggtgccc gaaagggaaa 1020
gtagacacag gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080
cgcaacgagc gcaacccttg ccaataattg ccatcattta gttgggcact ttattgggac 1140
tgccggtgac aaaccggagg aaggtgggga tgacgtcaag tcctcatggc ccttatggcc 1200
agggcttcac acgtaataca atggtcggta cagagggttg ccaacccgcg agggggagcc 1260
aatcccagaa agccgatcgt agtccggatt gcagtctgca actcgactgc atgaagtcgg 1320
aatcgctagt aatcgcggat cagcatgtcg cggtgaatac gttcccgggc cttgtacaca 1380
ccgcccgtca caccatggga gtgggttcta ccagaagtag ttagtctaac cgcaaggggg 1440
acgattacca cggtaggatt catgactggg gtgaagtcgt 1480

Claims (9)

1. Methylovorus (sp) HQ-1, accession number: cctccc NO: m2021252.
2. Use of the methylotrophic bacterium of claim 1 for the fermentative production of pyrroloquinoline quinone.
3. A composition comprising the methylotrophic bacterium HQ-1 of claim 1 and a fermentation broth having a pyrroloquinoline quinone concentration of 10.00mg/L or more.
4. A composition according to claim 3, wherein the concentration of pyrroloquinoline quinone in the fermentation broth is 40.00mg/L or more.
5. A method of producing pyrroloquinoline quinone, comprising: use of the methylotrophic bacterium HQ-1 of claim 1 for fermentative production of pyrroloquinoline quinone.
6. The method of claim 5, comprising:
activating the methyl-eating bacteria HQ-1 and inoculating the activated methyl-eating bacteria HQ-1 into a seed culture medium;
then inoculating the obtained seed liquid into a fermentation medium according to the volume ratio of 1-10% for fermentation culture to obtain pyrroloquinoline quinone.
7. The method of claim 6, comprising:
the activation is to culture in a slant culture medium for 12-24 hours at 25-32 ℃; and/or the number of the groups of groups,
the seed liquid is obtained by shaking culture in a seed culture medium at 25-32 ℃ and 120-220 rpm for 12-24 hours; and/or the number of the groups of groups,
the fermentation culture is carried out in a fermentation culture medium for 24-48 hours under the conditions of 25-32 ℃ and the rotating speed of 120-220 rpm.
8. The method of claim 6, wherein each 1L of the seed medium comprises: 3-10 g of ammonium sulfate, 5-20 g of methanol and KH 2 PO 4 1.5～3g、Na 2 HPO 4 ·12H 2 O 5～10g、MgSO 4 ·7H 2 0.1 to 0.5g of O, 0.01 to 0.05g of ferric citrate and CaCl 2 0.01～0.05g、MnCl 2 ·4H 2 O 0.1～0.5mg、ZnSO 4 ·7H 2 O 0.01～0.05g、CuSO 4 ·7H 2 0.1 to 0.5mg of O, 6.8 to 7.2 of pH and the balance of water.
9. The method of claim 6, wherein each 1L of the fermentation medium comprises: 3-20 g of nitrogen source, 5-20 g of methanol, 0.5-2 g of tyrosine, 0.5-2 g of glutamic acid and KH 2 PO 4 1.5～3g、Na 2 HPO 4 ·12H 2 O 5～10g、MgSO 4 ·7H 2 0.1 to 0.5g of O, 0.01 to 0.05g of ferric citrate and CaCl 2 0.01～0.05g、MnCl 2 ·4H 2 O 0.1～0.5mg、ZnSO 4 ·7H 2 O 0.01～0.05g、CuSO 4 ·7H 2 0.1 to 0.5mg of O, 6.8 to 7.2 of pH and the balance of water.
CN202110626696.9A 2021-06-04 2021-06-04 Methylophilus for producing pyrroloquinoline quinone and application thereof Active CN113337432B (en)

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CN113913448B (en) * 2021-07-23 2023-10-20 中国人民解放军军事科学院军事医学研究院 Method for improving yield of pyrroloquinoline quinone of methylotrophic bacteria and application

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