CN113337433A - Pseudomonas capable of producing pyrroloquinoline quinone and application thereof - Google Patents
Pseudomonas capable of producing pyrroloquinoline quinone and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to pseudomonas and a method for producing pyrroloquinoline quinone through fermentation of the pseudomonas. The invention screens a new strain Pseudomonas sp from soil near a pharmaceutical factory, which is named HQ-2, and the preservation number of the strain is as follows: CCTCC NO: m2021251. The invention also relates to application of pseudomonas (pseudomonas sp) HQ-2 in fermentation production of pyrroloquinoline quinone, a method for preparing pyrroloquinoline quinone and a composition. The strain is aerobically cultured for 1-2 days in a culture medium taking cane sugar and glucose as carbon sources, and the yield of pyrroloquinoline quinone can reach 60mg/L at the level of a shake flask. The strain screened by the invention can secrete PQQ to the outside of cells, has the characteristics of rapid growth and utilization of sucrose and glucose compared with the common PQQ producing strain, and provides a new strain for industrial fermentation production of PQQ.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to pseudomonas capable of producing pyrroloquinoline quinone and application thereof.
Background
Pyrroloquinoline quinone (PQQ, also known as Methoxatin) is a water-soluble quinone compound that is thermostable, first identified in bacteria as a cofactor for alcohol and glucose dehydrogenases, a third prosthetic group found in bacterial dehydrogenases, following flavin and nicotinamide nucleotides, and is referred to in the world medical community as the fourteenth vitamin. PQQ, as a novel water-soluble vitamin, is an oxidoreductase prosthetic group, is very rare, exists in some microorganisms, plants and animal tissues, and the phenomenon of low reproductive capacity and the like can occur in laboratory mice lacking the substance. Studies have shown that PQQ is present in everyday vegetables and fruits such as natto, celery, and kiwi fruit. The substance is present in viscera and body fluid of human body, and has a content of 0.8-5.9ng/g, wherein spleen has a highest content of 5.9 ng/g. The content of PQQ and derivatives thereof in human milk is up to 140-180mg/L, which is dozens of times higher than that of PQQ which is a common food, thus proving that the substance can play a vital role in the growth and development of newborn infants, and in addition, the content of PQQ in human milk is 50 times higher than that of animal and plant milk such as milk. It is generally accepted that PQQ is only synthesized in certain gram-negative bacteria, and PQQ is not synthesized in plants and animals and is obtained mainly by the dietary route.
Research shows that the PQQ has the functions of resisting oxidation, whitening skin, promoting amino acid absorption, preventing and treating senile dementia, conditioning neurological 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, i.e., chemical synthesis and microbial fermentation. The production of PQQ is realized by a chemical synthesis method firstly, the PQQ with the purity of about 97 percent can be obtained only by 16 process steps including 11 steps of chemical synthesis reaction and 5 steps of separation and purification in the prior PQQ chemical synthesis method, the chemical synthesis process is complex, a plurality of byproducts are produced, the purification technology is difficult, and the synthesis cost is high. Compared with the chemical synthesis method of PQQ, the microbial fermentation method is gradually a new research direction, has the advantages of environmental protection, low cost, few steps, easy separation and the like, and has the problems of lack of excellent industrial production strains, immature fermentation strategies and the like.
At present, PQQ is found in various strains, the maximum yield of the PQQ obtained by fermentation which is reported at present can reach 2g/L, but most of the reported yields are not high, so that the screening of high-yield strains is still the key for industrial production of PQQ.
The invention patent of China with publication number CN 104328155B, which utilizes Gluconobacter oxydans to produce pyrroloquinoline quinone, has PQQ yield up to 125mg/L through culture, but the culture time of the strain is 5 days, which is relatively long, and the realization of industrial mass production has certain difficulty.
The chinese patent publication No. CN 103740780B, "a method for synthesizing pyrroloquinoline quinone by lactic acid bacteria fermentation", obtains PQQ by primary culture, secondary culture, and fermentative anaerobic culture, has problems of complicated process and long time for preparing PQQ, and is difficult to apply in industrial mass production.
The invention discloses a Chinese patent with publication number CN 103898011B, namely a methylotrophic bacterium and a method for producing pyrroloquinoline quinone by fermenting the methylotrophic bacterium, wherein the yield of PQQ reaches 50-113 mg/L by aerobic culture for 3-4 days, but the strain takes methanol as a carbon source, so that the risk of methanol flammability and explosiveness and methanol residue in products exist.
The Chinese patent of invention with publication number CN106755169A discloses a method for increasing the yield of pyrroloquinoline quinone, wherein the fermentation strain is a fermentation strain of Pseudomonas aeruginosa, but the PQQ content is 30.96mg/L at most, and the carbon source is methanol of 7-50 g/L.
The master 'study on the synthesis of pyrroloquinoline quinone by Gluconobacter oxydans' conducted fermentation using Gluconobacter oxydans using 50g/L of glucose as a carbon source, but the PQQ production was low, up to 0.813 mg/L.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides pseudomonas (pseudomonas. sp) HQ-2 which is preserved in China center for type culture Collection (CCTCC for short) at 03-19 th 2021, wherein the preservation address is Wuhan university in Wuhan, China, and the preservation number is CCTCC NO: m2021251. The invention also provides a composition of pseudomonas and fermentation liquor, and a method for producing PQQ by fermentation.
The strain obtained by the invention is a strain of pseudomonas HQ-2(Pseudomonas sp) separated and screened from soil near a pharmaceutical factory, the growth cycle is about 1-2 days, the strain has the advantages of high growth speed and capability of utilizing cane sugar and glucose, and the yield can reach 60mg/L through preliminary optimization of a carbon nitrogen source and HPLC verification.
Specifically, the technical scheme of the invention is as follows:
1. pseudomonas sp HQ-2 with the deposit number: CCTCC NO: m2021251.
2. Pseudomonas HQ-2 according to item 1, the 16s rRNA gene sequence is shown in SEQ ID NO:1 (GCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGTCCTTCGGGATGCCGGCGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGACGCTAATACCGCATACGTCCTACGGGAGAAAGCGGGGGATCTTCGGACCTCGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCATTGACCTAATACGTCAGTGTTTTGACGTTACCAACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCAGCAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTACTGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGCTCCTTGAGAGCTTAGTGGCGCAGCTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCA).
3. Use of Pseudomonas HQ-2 according to item 1 or item 2 for the fermentative production of pyrroloquinoline quinone.
4. A composition comprising the Pseudomonas HQ-2 of item 1 or item 2 and a fermentation broth, wherein the concentration of pyrroloquinoline quinone in the fermentation broth is 5.00mg/L or more, specifically 10.00mg/L or more, 20.00mg/L or more, 30.00mg/L or more, 40.00mg/L or more, 50.00mg/L or more, 60.00mg/L or more, and preferably 60.00mg/L or more.
5. The composition of item 4, wherein the fermentation medium for producing the fermentation broth comprises a carbon source, a nitrogen source, and inorganic salts, wherein the carbon source is one or a combination of two of glucose and sucrose.
6. A method of producing pyrroloquinoline quinone, comprising: use of the pseudomonad HQ-2 of item 1 or item 2 for fermentation production of pyrroloquinoline quinone.
7. The method of item 6, comprising:
activating the pseudomonas HQ-2 and then inoculating the activated pseudomonas HQ-2 to a seed culture medium;
then inoculating the obtained seed liquid into a fermentation culture medium according to the volume ratio of 1-10% for fermentation culture to obtain pyrroloquinoline quinone.
8. The method of item 7, wherein,
the activation is carried out in a slant culture medium at the temperature of 25-32 ℃ for 12-24 hours; and/or the presence of a gas in the gas,
the seed solution is obtained by shake culture for 12-24 hours in a seed culture medium at 25-32 ℃ and 120-220 rpm; and/or the presence of a gas in the gas,
the fermentation culture is carried out in a fermentation culture medium at the temperature of 25-32 ℃ and the rotating speed of 120-220 rpm for 24-48 hours.
9. The method of item 7, wherein each 1L of the seed medium comprises: 3-10 g of ammonium sulfate, 5-20 g of methanol and KH2PO4 1.5～3g、Na2HPO4·12H2O 5～10g、MgSO4·7H20.1-0.5 g of O, 0.01-0.05 g of ferric citrate, CaCl2 0.01～0.05g、MnCl2·4H2O 0.1～0.5mg、ZnSO4·7H2O 0.01～0.05g、CuSO4·7H20.1-0.5 mg of O, 6.8-7.2 of pH and the balance of water; and/or the presence of a gas in the gas,
every 1L of the fermentation medium comprises: 3-20 g of nitrogen source, 2-30 g of carbon source, 0.5-2 g of tyrosine, 0.5-2 g of glutamic acid and KH2PO4 1.5～3g、Na2HPO4·12H2O 5～10g、MgSO4·7H20.1-0.5 g of O, 0.01-0.05 g of ferric citrate, CaCl2 0.01～0.05g、MnCl2·4H2O 0.1～0.5mg、ZnSO4·7H2O 0.01～0.05g、CuSO4·7H20.1-0.5 mg of O, 6.8-7.2 of pHs and the balance of water.
10. A food, cosmetic or pharmaceutical product comprising the composition of item 4 or 5 or pyrroloquinoline quinone produced by the method of any one of items 6 to 9.
The growth cycle of the pseudomonas (pseudomonas. sp) HQ-2 provided by the invention is about 1-2 days, and compared with the common PQQ producing bacteria, the pseudomonas (pseudomonas. sp) HQ-2 has the advantages of rapid growth and capability of utilizing cane sugar and glucose, and can avoid the risks of flammability and explosiveness and methanol residue in products when methanol is used. Through preliminary optimization of carbon and nitrogen sources and HPLC verification, the yield of PQQ can reach 60mg/L at the level of a shake flask.
Biological material preservation
The preservation date is as follows: 2021, 03 month and 19 days
The preservation unit: china Center for Type Culture Collection (CCTCC)
The preservation number is: CCTCC NO: m2021251
Drawings
FIG. 1: a cell morphology feature picture of pseudomonas (pseudomonas. sp) HQ-2;
FIG. 2: photographs of colony morphology of Pseudomonas (Pseudomonas sp) HQ-2;
FIG. 3: HQ-2 culture and PQQ standard HPLC results;
FIG. 4: the result of sequencing the 16s rRNA gene of Pseudomonas sp HQ-2.
Detailed Description
The following embodiments of the present invention are merely illustrative of specific embodiments for carrying out the present invention and should not be construed as limiting the present invention. Other changes, modifications, substitutions, combinations, and simplifications which may be made without departing from the spirit and principles of the invention are intended to be equivalents thereof and to fall within the scope of the invention.
As a specific embodiment of the present invention, the present invention relates to pseudomonas (pseudomonas. sp) HQ-2, which has been deposited in the chinese typical culture collection (CCTCC) at 03/19 of 2021, with the deposition address of wuhan, wuhan university, zip code: 430072, preservation number is CCTCC NO: m2021251, which can be used for the fermentative production of pyrroloquinoline quinone.
Pyrroloquinoline quinone, also known as methoxatin for short as PQQ, has a molecular formula of C14H6N2O8Molecular weight is 330.206, CAS registry number is 72909-34-3, a chemical intermediate. PQQ is a novel redox prosthetic group, and has various physiological functions of resisting oxidation, regulating the level of free radicals in vivo, promoting the regeneration of nerve growth factors and the like.
The 16s rRNA gene sequence of Pseudomonas HQ-2(Pseudomonas sp) is SEQ ID NO:1, see FIG. 4.
rRNA has the characteristics of uniqueness, importance and the like, has a special and conserved structure, and therefore has resistance to mutation affecting the structure. And the 16s rRNA gene has a polymorphic region between species, so that the sequence analysis can determine the evolutionary distance and the interrelation of various bacteria and identify the bacteria, and the sequence is ubiquitous in the bacteria, so that the method is suitable for the analysis of all bacteria.
The pseudomonas is a straight or slightly bent gram-negative bacillus and is a non-nuclear bacterium which moves with polar flagella and does not form spores. It is widely distributed in nature, such as soil, water, food, and air. There are capsules, flagella and pili.
The pseudomonas HQ-2 is obtained by separating and screening pseudomonas HQ-2 from soil near a pharmaceutical factory, the growth cycle is about 1-2 days, the pseudomonas HQ-2 has the advantages of high growth speed and capability of utilizing cane sugar and glucose, and the yield of the PQQ can reach 60mg/L at the level of a shake flask through preliminary optimization of a carbon-nitrogen source and HPLC verification.
As a specific embodiment of the invention, the pseudomonas HQ-2 obtained by the invention is used for producing pyrroloquinoline quinone by fermentation.
As a specific embodiment of the present invention, a composition obtained by fermentation of Pseudomonas HQ-2 of the present invention comprises the above Pseudomonas HQ-2 and a fermentation liquid, wherein the concentration of pyrroloquinoline quinone in the fermentation liquid is as high as 5.00mg/L or more, specifically 10.00mg/L or more, 15.00mg/L or more, 20.00mg/L or more, 25.00mg/L or more, 30.00mg/L or more, 35.00mg/L or more, 40.00mg/L or more, 45.00mg/L or more, 50.00mg/L or more, 55.00mg/L or more, or 60.00mg/L or more, preferably 60.00mg/L or more.
In a specific embodiment, the fermentation medium for producing the fermentation broth comprises a carbon source, a nitrogen source and inorganic salts, wherein the carbon source can be one or a combination of two of glucose and sucrose, and the nitrogen source can be one or a combination of more than two of peptone, yeast powder, soybean peptide, ammonium sulfate, beef extract and corn flour.
In a specific embodiment, the invention relates to a method for producing pyrroloquinoline quinone by fermentation, which comprises producing pyrroloquinoline quinone by using the above-mentioned pseudomonas HQ-2.
As a specific embodiment of the present invention, relates to a method for producing pyrroloquinoline quinone, which comprises: the pseudomonas HQ-2 is used for producing pyrroloquinoline quinone by fermentation.
In a specific embodiment, the pseudomonas HQ-2 is activated and then inoculated into a seed culture medium; then inoculating the obtained seed liquid into a fermentation culture medium according to the volume ratio of 1-10% (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 at 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).
In the present invention, each 1L of the slant culture medium contains 3-10 g of ammonium sulfate, 5-20 g of methanol, and KH2PO4 1.5～3g、Na2HPO4·12H2O 5～10g、MgSO4·7H20.1-0.5 g of O, 0.01-0.05 g of ferric citrate, CaCl2 0.01～0.05g、MnCl2·4H2O 0.1～0.5mg、ZnSO4·7H2O 0.01～0.05g、CuSO4·7H20.1-0.5 mg of O, 6.8-7.2 of pH and the balance of water.
In a specific embodiment, the seed solution is obtained by shake-culturing in a seed culture medium at 25 to 32 ℃ (specifically, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃ or 31 ℃), 120 to 220rpm (specifically, 130rpm, 140rpm, 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm or 210rpm) 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).
In a specific embodiment, the fermentation culture is performed in a fermentation medium under conditions of 25 to 32 ℃ (specifically, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃ or 31 ℃), 120 to 220rpm (specifically, 130rpm, 140rpm, 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm or 210rpm) 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).
When the pseudomonas (pseudomonas. sp) HQ-2 provided in this example is fermented to produce PQQ, the growth cycle is about 1 to 2 days, and PQQ is produced more rapidly than general PQQ-producing bacteria.
In a specific embodiment, each 1L of said seed medium comprises: 3-10 g of ammonium sulfate, 5-20 g of methanol and KH2PO4 1.5～3g、Na2HPO4·12H2O 5～10g、MgSO4·7H20.1-0.5 g of O, 0.01-0.05 g of ferric citrate, CaCl2 0.01～0.05g、MnCl2·4H2O 0.1～0.5mg、ZnSO4·7H2O 0.01～0.05g、CuSO4·7H20.1-0.5 mg of O, 6.8-7.2 of pH and the balance of water.
The seed culture medium is used for spore germination, growth and mass propagation of thallus, and makes the thallus grow stout and become a 'seed' with strong activity.
In a specific embodiment, each 1L of said fermentation medium comprises: 3-20 g of nitrogen source, 2-30 g of carbon source, 0.5-2 g of tyrosine, 0.5-2 g of glutamic acid and KH2PO4 1.5～3g、Na2HPO4·12H2O 5～10g、MgSO4·7H20.1-0.5 g of O, 0.01-0.05 g of ferric citrate, CaCl2 0.01～0.05g、MnCl2·4H2O 0.1～0.5mg、ZnSO4·7H2O0.01～0.05g、CuSO4·7H20.1-0.5 mg of O, 6.8-7.2 of pHs and the balance of water.
The fermentation medium is used for the growth, propagation and synthesis of the strain. It not only ensures that the seeds can grow rapidly after being inoculated to reach a certain hypha concentration, but also ensures that the grown bacteria can rapidly synthesize the required products.
The carbon source is a general name of a type of nutrient substance which contains carbon and can be utilized by the growth and reproduction of microorganisms. The carbon source has the effect on the growth and metabolism of bacteria, and mainly provides a carbon skeleton of cells, provides energy required by the vital activities of the cells, and provides a carbon skeleton for synthesizing products. The carbon source plays an important role in preparing a bacteria culture medium and provides a material basis for the normal growth and division of bacteria. Wherein, the carbon source can be one or the combination of two of glucose and sucrose.
A nitrogen source is required for bacterial growth and product synthesis. Nitrogen sources are mainly used for the synthesis of bacterial cell substances (amino acids, proteins, nucleic acids, etc.) and nitrogen-containing metabolites. The nitrogen source used in the fermentation medium in the present application includes an organic nitrogen source and/or an inorganic nitrogen source. Wherein the nitrogen source can be one or more of peptone, yeast powder, soybean peptide, ammonium sulfate, beef extract, and corn flour.
In a specific embodiment, there is disclosed a food, cosmetic or pharmaceutical product comprising the above composition or the pyrroloquinoline quinone produced by the above method.
Food refers to various finished products and raw materials for people to eat or drink and the products which are food and traditional Chinese medicinal materials according to the tradition, but do not include the products for treating. The cosmetic is a chemical industrial product or a fine chemical product which is applied to any part of the surface of a human body, such as skin, hair, nails, lips and teeth, by smearing, spraying or the like, so as to achieve the purposes of cleaning, maintaining, beautifying, decorating and changing the appearance, or correcting the odor of the human body and keeping a good state.
The medicine is a substance for preventing, treating and diagnosing human diseases, purposefully regulating the physiological functions of human and prescribing indications or functional indications, administration and dosage, and comprises traditional Chinese medicines, chemical medicines, biological products and the like.
The fermentation medium provided by the embodiment can utilize sucrose and glucose as carbon sources, the conditions that the fermentation medium is inflammable and explosive when methanol is used and the risk of methanol residue in a product can be avoided, and when pseudomonas (pseudomonas. sp) HQ-2 is used for fermenting and producing PQQ by using the fermentation medium, the yield of the PQQ can reach 60mg/L at the level of a shake flask.
Examples
The following experimental methods are all conventional methods unless otherwise specified.
The materials, reagents and the like used below are commercially available unless otherwise specified.
Screening a culture medium: each 1L of the screening medium contains 3g of ammonium sulfate, 10g of methanol, KH2PO41.5 g of Na2HPO 4.12H 2O 7.67.67 g, 0.2g of MgSO 4.7H 2O 0.2, 0.02g of ferric citrate, 20.03 g of CaCl, 0.2mg of MnCl 2.4H 2O 0.2, 0.02g of ZnSO 4.7H 2O 0.02, 0.2mg of CuSO 4.7H 2O 0.2, pH 7.0 and the balance of water.
Seed culture medium: consistent with the composition of the screening medium.
Fermentation medium: each 1L of the fermentation medium contains nitrogen source, carbon source, tyrosine 1.5g, glutamic acid 1.5g, KH2PO4 1.4g、Na2HPO4·12H2O 7.67g、MgSO4·7H20.2g of O, 0.02g of ferric citrate and CaCl20.03g、MnCl2·4H2O 0.2mg、ZnSO4·7H2O 0.02g、CuSO4·7H2O0.2 mg, pH 7.0, and the balance water.
Example 1 screening of PQQ-producing bacteria
Collecting 61 parts of soil sample or water sample from Shandong, Henan and inner Mongolia areas, enriching by a screening culture medium, diluting and coating on a screening culture medium plate, and culturing at 30 ℃ for 3-5 days to obtain a primary screening strain. Respectively inoculating the primary screened strains into test tubes filled with 5mL of screening culture medium, carrying out shake culture at 30 ℃ for 2 days, then transferring the primary screened strains into a triangular flask filled with 50mL of screening culture medium, and fermenting at 30 ℃ and 200rpm for 3-5 days. As shown in Table 1, the PQQ content in the fermentation supernatant was precisely measured by HPLC, wherein the highest yield of 22.32mg/L was obtained for the strain numbered Pseudomonas spHQ-2.
TABLE 1 different isolates producing PQQ
| 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-20 | 10.45 |
| HQ-4 | 12.03 | HQ-21 | 20.14 |
| HQ-5 | 3.26 | HQ-22 | 13.45 |
| HQ-6 | 2.87 | HQ-23 | 11.08 |
| HQ-7 | 17.65 | HQ-24 | 1.35 |
Example 2 morphological observations
The morphological characteristics of Pseudomonas (Pseudomonas sp) HQ-2 are shown in figure 1. Specifically, the cells were observed to be short rods by using an OLYMPUS CX33 microscope.
The colony morphology of Pseudomonas (Pseudomonas sp) HQ-2 is shown in figure 2. The colonies were observed to be off-white, flat, wet, round.
Example 3 Strain identification
The genome of the strain is sequenced and identified by the company Limited in Biotechnology engineering (Shanghai), and the determination result of the 16s rRNA gene sequence of the pseudomonas HQ-2 is shown in SEQ ID NO. 1 (see figure 4 in detail).
EXAMPLE 4 detection of product PQQ
PQQ measurement was performed by high performance liquid chromatography using the fermentation broth numbered pseudomonas. sp HQ-2 in example 1 and a PQQ standard as test samples, and the HPLC conditions were: the sample volume was 10 μ L, the column temperature was 40 ℃, the flow rate was 0.6mL/min, the detection wavelength was 252nm, the column temperature was 85:15 ℃, and the mobile phase (0.1% trifluoroacetic acid: acetonitrile): eclips plus C18, the peaks of the HQ-2 fermentation broth and the PQQ standard were completely consistent by HPLC comparison, as shown in FIG. 3.
Example 5 carbon Source optimization of fermentation Medium
Strain activation: pseudomonas sp HQ-2 was inoculated into a slant medium (the composition was the same as that of the above seed medium), and cultured at 30 ℃ for 24 hours.
Fermentation culture: inoculating activated Pseudomonas sp HQ-2 strain into seed culture medium, shake culturing at 30 deg.C and 200rpm for 20 hr, inoculating seed solution 5% (v/v) into triangular flask containing fermentation medium (nitrogen source: ammonium sulfate 3g/L), containing 50mL of 250mL triangular flask, and fermenting at 30 deg.C and 200rpm for 2 days. Wherein glucose, methanol, sucrose, fructose, lactose, glycerol and maltose are respectively used as carbon sources of the fermentation medium (the content of the carbon source is 10 g/L). Determination of Biomass (OD) after completion600) Meanwhile, NBT-Gly chemistry was used to rapidly determine the PQQ content in the fermentation supernatant, and the results are shown in Table 2.
TABLE 2 carbon source optimization results
| Carbon source | PQQ yield (mg/L) | OD600 |
| Sucrose | 35.05 | 3.37 |
| Glucose | 17.18 | 3.80 |
| Methanol | 20.13 | 2.82 |
| Fructose | 0 | 3.81 |
| Lactose | 0 | 0.32 |
| Glycerol | 0 | 1.43 |
| Maltose | 0 | 0.43 |
Example 6 Nitrogen source optimization of fermentation Medium
Strain activation: the original strain was inoculated into a slant medium (having the same composition as the above-mentioned seed medium), and cultured at 30 ℃ for 24 hours.
Fermentation culture: inoculating activated Pseudomonas sp HQ-2 strain to seed culture medium, shake culturing at 32 deg.C and 200rpm for 24 hr, and inoculating the seed solution to fermentation medium at 3% (v/v)In a triangular flask containing 250mL of a culture medium (containing 10g/L of sucrose and 5g/L of glucose as carbon sources), 50mL of a solution is fermented at 32 ℃ and 200rpm for 2 days, wherein peptone, yeast powder, soybean peptide, ammonium sulfate, beef extract and corn flour are respectively used as nitrogen sources (the nitrogen source is 3g/L) of the fermentation medium. Determination of Biomass (OD) after completion600) Meanwhile, NBT-Gly chemistry was used to rapidly determine the PQQ content in the fermentation supernatant, and the results are shown in Table 3.
TABLE 3 Nitrogen source optimization results
| Nitrogen source | PQQ yield (mg/L) | OD600 |
| Peptone | 5.14 | 11.93 |
| Yeast powder | 2.38 | 10.45 |
| Soybean peptide | 60.00 | 13.67 |
| Ammonium sulfate | 41.82 | 3.93 |
| Beef extract | 10.18 | 10.34 |
| Corn flour | 13.05 | 13.15 |
While embodiments of the present application have been described above, the present application is not limited to the specific embodiments and applications described above, which are intended to be illustrative, instructive, and not limiting. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto and changes may be made without departing from the scope of the invention as defined by the appended claims.
Sequence listing
<110> Huaxi Biotechnology Ltd
<120> pseudomonas capable of producing pyrroloquinoline quinone and application thereof
<130> TPE01430
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<170> PatentIn version 3.5
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<213> Artificial sequence
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<223> artificially synthesized
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cgggatgccg gcgagcggcg gacgggtgag taatgcctag gaatctgcct ggtagtgggg 120
gataacgttc ggaaacggac gctaataccg catacgtcct acgggagaaa gcgggggatc 180
ttcggacctc gcgctatcag atgagcctag gtcggattag ctagttggtg gggtaatggc 240
tcaccaaggc gacgatccgt aactggtctg agaggatgat cagtcacact ggaactgaga 300
cacggtccag actcctacgg gaggcagcag tggggaatat tggacaatgg gcgaaagcct 360
gatccagcca tgccgcgtgt gtgaagaagg tcttcggatt gtaaagcact ttaagttggg 420
aggaagggca ttgacctaat acgtcagtgt tttgacgtta ccaacagaat aagcaccggc 480
taacttcgtg ccagcagccg cggtaatacg aagggtgcaa gcgttaatcg gaattactgg 540
gcgtaaagcg cgcgtaggtg gttcagcaag ttggatgtga aagccccggg ctcaacctgg 600
gaactgcatc caaaactact gagctagagt acggtagagg gtggtggaat ttcctgtgta 660
gcggtgaaat gcgtagatat aggaaggaac accagtggcg aaggcgacca cctggactga 720
tactgacact gaggtgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 780
cgccgtaaac gatgtcgact agccgttggg ctccttgaga gcttagtggc gcagctaacg 840
cgataagtcg accgcctggg gagtacggcc gcaaggttaa aactcaaatg aattgacggg 900
ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttacctg 960
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gcaacccttg tccttagtta ccagca 1106
Claims (10)
1. Pseudomonas sp HQ-2 with the deposit number: CCTCC NO: m2021251.
2. The Pseudomonas HQ-2 of claim 1, wherein the 16s rRNA gene sequence is shown in SEQ ID NO. 1.
3. Use of pseudomonad HQ-2 according to claim 1 or 2 for the fermentative production of pyrroloquinoline quinone.
4. A composition comprising pseudomonas HQ-2 as described in claim 1 or 2 and a fermentation broth, wherein the concentration of pyrroloquinoline quinone in the fermentation broth is 5.00mg/L or more, preferably 60.00mg/L or more.
5. The composition of claim 4, wherein the fermentation medium used to produce the fermentation broth comprises a carbon source, a nitrogen source, and inorganic salts, wherein the carbon source is one or a combination of glucose and sucrose.
6. A method of producing pyrroloquinoline quinone, comprising: use of pseudomonas HQ-2 according to claim 1 or 2 for the fermentative production of pyrroloquinoline quinone.
7. The method of claim 6, comprising:
activating the pseudomonas HQ-2 and then inoculating the activated pseudomonas HQ-2 to a seed culture medium;
then inoculating the obtained seed liquid into a fermentation culture medium according to the volume ratio of 1-10% for fermentation culture to obtain pyrroloquinoline quinone.
8. The method of claim 7, wherein,
the activation is carried out in a slant culture medium at the temperature of 25-32 ℃ for 12-24 hours; and/or the presence of a gas in the gas,
the seed solution is obtained by shake culture for 12-24 hours in a seed culture medium at 25-32 ℃ and 120-220 rpm; and/or the presence of a gas in the gas,
the fermentation culture is carried out in a fermentation culture medium at the temperature of 25-32 ℃ and the rotating speed of 120-220 rpm for 24-48 hours.
9. The method of claim 7,
each 1L of the seed culture medium contains: 3-10 g of ammonium sulfate, 5-20 g of methanol and KH2PO41.5～3g、Na2HPO4·12H2O 5～10g、MgSO4·7H20.1-0.5 g of O, 0.01-0.05 g of ferric citrate, CaCl20.01～0.05g、MnCl2·4H2O 0.1～0.5mg、ZnSO4·7H2O 0.01～0.05g、CuSO4·7H20.1-0.5 mg of O, 6.8-7.2 of pH and the balance of water; and/or the presence of a gas in the gas,
every 1L of the fermentation medium contains: 3-20 g of nitrogen source, 2-30 g of carbon source, 0.5-2 g of tyrosine, 0.5-2 g of glutamic acid and KH2PO41.5～3g、Na2HPO4·12H2O 5～10g、MgSO4·7H20.1-0.5 g of O, 0.01-0.05 g of ferric citrate, CaCl20.01～0.05g、MnCl2·4H2O 0.1～0.5mg、ZnSO4·7H2O 0.01～0.05g、CuSO4·7H20.1-0.5 mg of O, 6.8-7.2 of pH and the balance of water.
10. A food, cosmetic or pharmaceutical product comprising the composition of claim 4 or 5 or pyrroloquinoline quinone produced by the method of any one of claims 6 to 9.
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| 钟杉杉: "吡咯喹啉醌高产菌的筛选、诱变、发酵及基因克隆", 《中国优秀博硕士学位论文全文数据库(硕士) 基础科学辑(月刊)》 * |
| 钟杉杉等: "吡咯喹啉醌生产菌的发酵条件优化", 《北京化工大学学报(自然科学版)》 * |
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