CN118006517B - CO (carbon monoxide)2Methyltrophic bacillus for autotrophic production of pyrroloquinoline quinone and application thereof - Google Patents
CO (carbon monoxide)2Methyltrophic bacillus for autotrophic production of pyrroloquinoline quinone and application thereof Download PDFInfo
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- MMXZSJMASHPLLR-UHFFFAOYSA-N pyrroloquinoline quinone Chemical compound C12=C(C(O)=O)C=C(C(O)=O)N=C2C(=O)C(=O)C2=C1NC(C(=O)O)=C2 MMXZSJMASHPLLR-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 54
- 230000001651 autotrophic effect Effects 0.000 title claims abstract description 19
- 238000004519 manufacturing process Methods 0.000 title abstract description 8
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 title 1
- 229910002091 carbon monoxide Inorganic materials 0.000 title 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 81
- 238000000034 method Methods 0.000 claims abstract description 13
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- 239000002609 medium Substances 0.000 claims description 12
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- 239000007836 KH2PO4 Substances 0.000 claims description 3
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- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 claims description 3
- 239000011565 manganese chloride Substances 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 235000013619 trace mineral Nutrition 0.000 claims description 3
- 239000011573 trace mineral Substances 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
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- 239000011686 zinc sulphate Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 2
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of microorganisms, and particularly relates to a methylotrophic bacillus for autotrophic production of pyrroloquinoline quinone by CO 2 and application thereof. The invention provides a methylotrophic bacillus for autotrophic production of pyrroloquinoline quinone by CO 2, which is methylotrophic bacillus MB-2 (Methylobacterium), and has the preservation number of: CGMCC No.29909. The method can efficiently utilize methanol, autotrophy CO 2 and produce pyrroloquinoline quinone, and has a high application value.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a methylotrophic bacillus for autotrophic production of pyrroloquinoline quinone by CO 2 and application thereof.
Background
Over the past decade scientists have been motivated to find a new renewable resource for chemical production due to insufficient petroleum fuels and global warming caused by greenhouse gas emissions (e.g., carbon dioxide). Glucose is the most abundant monosaccharide in nature, is a basic energy source for cells, and is also an important raw material for the biorefinery industry. At present, a plant-biomass-sugar route is dominant in glucose production, but the route faces the problems of long plant growth period, large biomass collection radius, high pretreatment process cost and the like.
Aiming at the increasingly serious global climate crisis and food shortage problem, the development of a high-efficiency sustainable autotrophic synthetic route of carbon dioxide has important significance; when CO 2 gas is utilized by an autotrophic system, CO 2 has the potential to be a renewable carbon source. The ability of autotrophic microorganisms to fix and convert CO 2 to biomass and potentially valuable products is of particular interest in the green production field in academia and industry. Autotrophic microorganisms require an energy source, which may be light (photoautotrophic) or an inorganic electron donor (fossil autotrophic), to incorporate CO 2 into the biomass. Autotrophic refineries are considered as a more sustainable use of carbon and an energy efficient alternative to heterotrophic culture for converting organic carbon materials into similar products. Therefore, development of CO 2 autotrophic microorganisms with application value is needed.
In addition, pyrroloquinoline quinone (pyrroloquinoline quinone, PQQ) is a 3 rd oxidoreductase coenzyme found following pyridine nucleotide [ NAD (P) +] and riboflavin (FMN, FAD) as a coenzyme for methanol dehydrogenase in methylotrophic bacteria, and participates in the redox process. The PQQ is the only active small molecule which exists in all animal and plant tissues and participates in growth and development so far, has physiological functions of promoting growth, optimizing mitochondrial biogenesis, scavenging free radicals, enhancing cell metabolism, improving stress resistance and the like, and has important development prospect in the fields of food, medicine and health care.
The strain which naturally produces PQQ in large quantities is mainly derived from methylotrophic bacillus (Methylobacterium), rhizoctonia (Hyphomicrobium), paracoccus (Paracoccus), gluconobacter (Gluconobacter), pseudomonas (Pseudomonas) and the like.
Disclosure of Invention
The invention provides a method for screening a bacterial strain capable of efficiently utilizing methanol and autotrophic CO 2 from chemical and textile wastewater, wherein ITS is identified as methylotrophic bacillus and named as methylotrophic bacillus MB-2. The strain can utilize sugar sources, methanol, glycerol, CO 2 and the like as the only carbon sources and can grow rapidly. The invention discovers that methylotrophic bacillus MB-2 (also called methylotrophic bacillus) can autotrophy CO 2 for the first time, and when 0.2 percent and 0.5 percent of methanol are added, the growth amount of the methylotrophic bacillus MB-2 is about 60 percent higher than that of the conventional methylotrophic bacillus, and the growth is derived from the autotrophy of CO 2. The protein content of the methylotrophic bacillus MB-2 is 63.5%, the amino acid composition is 57.2%, and the nutritive value is obvious. When methanol and CO 2 are used as carbon sources, the strain can produce 39.45 mg/L PQQ. Thus, the present invention has been completed.
The invention provides a methylotrophic bacillus for autotrophic production of pyrroloquinoline quinone by CO 2, which is methylotrophic bacillus MB-2 (Methylobacterium), and has the preservation number of: CGMCC No. 29909, which was preserved in China general microbiological culture Collection center (CGMCC) at 29 months of 2024, and is classified and named as methylobacterium Methylobacterium sp (Chinese name is also called methylotrophic bacillus) in North Star Xila No. 1, no. 3 of the Korean area of Beijing city.
The present invention further provides a method for culturing the methylotrophic bacillus, which comprises culturing the methylotrophic bacillus in a salt-free medium.
Preferably, the inorganic salt culture is delft inorganic salt culture medium, and no carbon source is added, or methanol is used as the only carbon source.
Specifically, the culture conditions are as follows: 28-32 deg.c and 600-1000 rpm. Preferably, the culture conditions are as follows: at 30℃at 800 rpm.
The invention provides application of the methylotrophic bacillus in CO 2 fixation and conversion into biomass.
The invention also provides application of the methylotrophic bacillus in preparation of pyrroloquinoline quinone.
The invention also provides a method for preparing pyrroloquinoline quinone by culturing the methylotrophic bacillus to produce pyrroloquinoline quinone.
In a specific embodiment, the culture medium is a PQQ fermentation culture medium, and the formula is as follows: yeast powder 1.0g/L, methanol 30.0g/L,MgSO4·7H2O 1.5g/L,(NH4)2SO4 3.0g/L,KH2PO4 1.4g/L,Na2HPO4 3.0g/L; trace elements: ferric citrate 0.3mg/L,CaCl2·2H2O 0.3mg/L,ZnSO4·7H2O 0.05mg/L,CuSO4·5H2O 0.5mg/L,CoCl2·6H2O 3.2mg/L,MnCl2·4H2O 0.5mg/L,pH 7.0.
More specifically, the culture temperature was 30 ℃.
Therefore, the invention provides the wild methylotrophic bacillus with high protein synthesis and CO 2 autotrophic microorganism, which can efficiently utilize methanol, autotrophy CO 2 and produce pyrroloquinoline quinone, and has a great application value.
Drawings
Fig. 1: strain identification analysis of MB 2.
Fig. 2: methylotrophic bacillus MB-2 plate colony map.
Fig. 3: CO2 is used as a carbon source, and the methylotrophic bacillus MB-2 is fermented and grown.
Fig. 4: methylotrophic bacillus MB-2 growth under CO2 or different methanol culture conditions.
Fig. 5: the color and the character of the bacillus methylotrophicus MB-2 are cultured by shaking the bacillus methylotrophicus MB-2.
Fig. 6: the color and the state of the methylotrophic bacillus MB-2 thallus.
Biological material preservation information
The methylotrophic bacillus MB-2 strain of the invention is preserved in China general microbiological culture collection center (CGMCC for short, address is North Star Xiyu No. 1 and No.3 in the Chaiyang area of Beijing city), and the preservation number is: CGMCC No.29909, the preservation time is: 2024, 02, 29. Its classification is designated methylobacterium Methylobacterium sp (chinese name also known as methylotrophic bacillus).
Detailed Description
The invention will be further illustrated by the following specific examples in order to provide a better understanding of the invention, but without limiting the invention thereto.
Example 1:
1. screening of methylotrophic bacillus MB-2
The invention takes a sample of waste water from Shandong Dongying chemical industry and textile industry, puts the sample into a triangular flask containing 95 mL sterile water and 10 glass beads, and oscillates 30 min at 30 ℃ and 180 rpm. Bacterial suspension 1 mL was subjected to 10 -1 -10-7 serial concentration gradient dilutions, then 10 -5、10 -6、10-7 dilutions were plated onto Delft medium plates with 0.5% methanol as the sole carbon source and incubated upside down at 30℃for 5d.
Purifying: after colonies were formed on a plate of medium with methanol as the sole carbon source, single colonies were picked up and cultured in a 96-well plate with 0.2% methanol as the sole carbon source at 30℃and 180rpm for 2 days, and OD600nm was measured.
Finally 8 strains which can grow by using methanol are screened, one of the strains can grow fastest in the presence of 0.5% methanol as the only carbon source, the OD600 can reach 6.38 after culturing for 30 hours, the strain is MB-2, and the obtained bacterial colony is stored at 4 ℃.
2. Identification of methylotrophic bacillus MB-2
The strain MB-2 was obtained by this screening, via a pair of known universal 16s primers:
16sF (5’ to3’): AGAGTTTGATCCTGGCTCA；
16SR (5’to3’): GGTTACCTTGTTACGACTT；
PCR amplification was performed, the amplified products were sequenced, and aligned and subjected to treelike analysis (FIG. 1). The results showed that this strain was identified as methylotrophic bacillus and named methylotrophic bacillus MB-2.
Plate culture and observation: methylotrophic bacillus MB-2 was cultured on YPD solid plates at 30℃for 3 days, with smooth colony surfaces and a pale pink color, as shown in FIG. 2.
Example 2:
delft inorganic salt culture medium
The liquid medium ：7.5 g（NH4）2SO4,14.4 g KH2PO4,0.5 g MgSO4·7H2O,pH was adjusted to 6.5 and sterilized at 121℃to 20: 20 min.
Culture method
The bacterial solution in the 5 ml test tube is centrifuged for one minute under the condition of 12000 r/min, the supernatant is discarded, the bacterial solution is resuspended by sterile water, and the steps are repeated for three times, and the OD600 of the seed solution is resuspended to 5.0 for standby.
Four parallel experiments were performed in 48 well plates with a methanol content gradient (0,0.2%, 0.5%,0.8%,1.1%, 1.4%) set and 1mL total of Delft inorganic salt broth and methanol was added to each loading well. After 3 uL bacterial solutions are added into each hole, the holes are placed in a full-automatic growth instrument for culture, the culture conditions are set to be 30 ℃, the rotation speed is 800 r/min, the OD600 detection time interval is 1h, and a growth curve is drawn.
Culture results
When using delft inorganic salt medium, without adding other carbon source, culturing methylotrophic bacillus MB-2, the rotation speed is low, and a stage of prolonged incubation period is obvious, and no bacterial growth is seen after culturing for 5 days. When using a rotational speed of 800 rpm, the high rotational speed facilitates the dissolution of CO 2 in air into the liquid medium as a carbon source, entering the logarithmic phase after 24 h culture. When cultured for 60 hours, the OD600 of the methylotrophic bacillus MB-2 can reach 3.0; the methylotrophic bacillus MB-2 has the autotrophic capacity of CO 2 and has high transformation efficiency. To verify the CO 2 autotrophic capacity of methylotrophic bacillus MB-2, in 3L fermentation culture, delft inorganic salt culture medium was used, no other carbon source was added for culture, aeration was only carried out, and after 30 hours of cultivation of methylotrophic bacillus MB-2 at 1000 revolutions per minute, the fermentation broth became turbid, demonstrating that methylotrophic bacillus MB-2 can grow normally using only CO 2 (FIG. 3).
When 0.2% methanol is added into delft inorganic salt culture medium, the methylotrophic bacillus MB-2 is cultured for 96 hours, and the OD600 can reach 4.91; when the concentration of methanol is continuously increased to 0.5%, the delay period of the thalli is obviously increased, but once the cells grow, the growth rate is not affected by methanol, and the OD600 can reach 6.38 after 196 hours of culture; when the methanol concentration was further increased to 0.8%,1.1% and 1.4%, the delay period of the growth of methylotrophic bacillus MB-2 was also further increased, and the cell amount was not increased with the increase of the methanol concentration, but the cell growth was inhibited (FIG. 4), indicating that the optimum methanol utilization concentration for the growth of the strain was 0.5%.
Purchase of methylotrophic bacillus (SHBCC C10397) when 0.2%,0.5% methanol was used for cultivation, and the accumulation amount of the bacterial OD600 only reached 3.2,4.6 under the same time conditions as that of methylotrophic bacillus MB-2; under the same culture conditions, the cumulative bacterial volume of the methylotrophic bacillus MB-2 is obviously about 60 percent higher than that of the conventional methylotrophic bacillus, and the partial growth is derived from the autotrophy of CO 2. The methylotrophic bacillus MB-2 has strong autotrophic capacity of CO 2, and has prospect in the aspects of fixing and converting CO 2 into biomass and potentially valuable products.
Example 3:
PQQ fermentation Medium (g/L): yeast powder 1.0, methanol 30.0,MgSO4·7H2O 1.5,(NH4)2SO4 3.0,KH2PO4 1.4,Na2HPO4 3.0; trace elements (mg/L): ferric citrate 0.3,CaCl2·2H2O 0.3,ZnSO4·7H2O 0.05,CuSO4·5H2O 0.5,CoCl2·6H2O 3.2,MnCl2·4H2O 0.5,pH 7.0
PQQ spectroscopy detection: methylotrophic bacillus MB-2 was cultured for 5 days at 30℃using PQQ fermentation, and the supernatant was centrifuged. Samples were transferred to 96 well shallow plates and the difference between OD 326 and OD 400 was detected using a microplate reader, while screening media was treated and detected under the same conditions, thereby subtracting media absorbance. The calculation results were averaged over 3 parallel fermentation experiments.
Determination of cell crude protein:
(1) Collecting thalli;
(2) Washing the cells 3 times with ddH 2 O to remove solid salts;
(3) Under the condition of sulfuric acid, the treated thalli carry out digestion reaction through thermocatalytic high-temperature oxidation reaction: in this way, even very stable, complex nitrogen-containing compounds can be digested in certain amounts; the sample directly enters a high-temperature area in the filled reaction tube, and the sample in the area undergoes high-temperature catalysis and oxidation reaction in carrier gas flow to generate NO; the pyrolyzed gas is cooled in a coil condenser, then the cooling water is separated from the measuring gas in the subsequent TIC condenser, and after further drying and removal of corrosive gases, the NO measuring gas is passed through a CLD detector; the concentration of nitrogen oxides is measured several times per second, and a peak graph of the change of the signal with time can be obtained;
(4) Peak area is proportional to the concentration of nitrogen in the measured solution;
(5) The nitrogen content in the sample can be calculated by using a previously determined standard curve;
(6) After the total nitrogen amount was measured, the calculation formula of the crude protein content in the cells was as follows, protein (g/100 g) =total nitrogen amount (g/100 g) ×6.25.
After fermentation of methylotrophic bacillus MB-2, pyrroloquinoline quinone formation was detected in the supernatant after centrifugation.
The methylotrophic bacillus MB-2PQQ fermentation medium was cultured at 30℃for 5 days, and the fermentation broth appeared pink when methanol and CO 2 were used as carbon sources (FIG. 5). After centrifugation, the supernatant of the fermentation broth was tested for PQQ production, and the strain produced 39.45 mg PQQ/L at a rate of 7.89mg/L/d. The sediment part is methylotrophic bacillus MB-2 thallus, the color of the thallus is reddish brown (figure 6), and through Kjeldahl nitrogen determination instrument and automatic analysis of amino acid, the crude protein content of the thallus is 63.5%, the amino acid composition is 57.2%, and the nutritive value is obvious.
Claims (10)
1. A CO 2 autotrophic pyrroloquinoline quinone-producing methylotrophic bacterium (Methylobacterium sp.) characterized by the deposit number: CGMCC No.29909.
2. A method for culturing the methylotrophic bacterium of claim 1, wherein the culturing is carried out in an inorganic salt medium.
3. The method of claim 2, wherein the inorganic salt medium is delft inorganic salt medium and no carbon source is added or methanol is the only carbon source.
4. A method according to claim 3, wherein the culture conditions are: 28-32 deg.c and 600-1000 rpm.
5. The method of claim 4, wherein the culturing conditions are: 30. 800 rpm.
6. Use of methylotrophic bacillus according to claim 1 for the fixation and conversion of CO 2 to biomass.
7. Use of methylotrophic bacillus according to claim 1 for the preparation of pyrroloquinoline quinone.
8. A method of preparing pyrroloquinoline quinone, wherein the methylotrophic bacillus of claim 1 is cultured to produce pyrroloquinoline quinone.
9. The method of claim 8, wherein the culture medium used for the cultivation is a PQQ fermentation medium comprising the following components: yeast powder 1.0g/L, methanol 30.0g/L,MgSO4·7H2O 1.5g/L,(NH4)2SO4 3.0g/L,KH2PO4 1.4g/L,Na2HPO4 3.0g/L; trace elements: ferric citrate 0.3mg/L,CaCl2·2H2O 0.3mg/L,ZnSO4·7H2O 0.05mg/L,CuSO4·5H2O 0.5mg/L,CoCl2·6H2O 3.2mg/L,MnCl2·4H2O 0.5mg/L,pH 7.0.
10. The method of claim 9, wherein the incubation temperature is 30 ℃.
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| CN117230133A (en) * | 2023-09-15 | 2023-12-15 | 广西天铭药业有限公司 | Method for improving pyrroloquinoline quinone titer of methylotrophic bacteria |
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