CN113308398A - Method for rapidly screening microorganisms capable of producing PQQ - Google Patents

Method for rapidly screening microorganisms capable of producing PQQ Download PDF

Info

Publication number
CN113308398A
CN113308398A CN202110555665.9A CN202110555665A CN113308398A CN 113308398 A CN113308398 A CN 113308398A CN 202110555665 A CN202110555665 A CN 202110555665A CN 113308398 A CN113308398 A CN 113308398A
Authority
CN
China
Prior art keywords
pqq
methanol
screening
enrichment culture
culture solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110555665.9A
Other languages
Chinese (zh)
Inventor
王玮
袁方
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Yiming Biological Science & Technology Co ltd
East China University of Science and Technology
Original Assignee
Jiangsu Yiming Biological Science & Technology Co ltd
East China University of Science and Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu Yiming Biological Science & Technology Co ltd, East China University of Science and Technology filed Critical Jiangsu Yiming Biological Science & Technology Co ltd
Priority to CN202110555665.9A priority Critical patent/CN113308398A/en
Publication of CN113308398A publication Critical patent/CN113308398A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention provides a method for rapidly screening microorganisms capable of producing PQQ, and relates to the fields of microbiology and biotechnology. The method for rapidly screening the PQQ-producing microorganism comprises the following steps: s1, enrichment culture of microbial flora: selecting a microbial flora, inoculating the microbial flora to a methanol enrichment culture solution, and carrying out enrichment culture to obtain an enrichment culture solution; s2, screening of microbial flora: and selecting an enrichment culture solution, coating the enrichment culture solution on a methanol screening flat plate, and continuously increasing the content of methanol to obtain the strain which rapidly grows by using the methanol. The invention provides a method for rapidly screening microorganisms capable of producing PQQ, which takes methanol as an antagonistic factor to carry out directional screening or domestication in a laboratory to breed a PQQ high-yield strain, and has simple operation and easy repetition.

Description

Method for rapidly screening microorganisms capable of producing PQQ
Technical Field
The invention relates to the field of microbiology and biotechnology, in particular to a method for rapidly screening microorganisms capable of producing PQQ.
Background
Pyrroloquinoline quinone, known under the english name of pyrroquinoline quinone (PQQ), was first discovered in 1964 as a cofactor for many bacterial dehydrogenases (methanol dehydrogenase, ethanol dehydrogenase, glucose dehydrogenase, etc.), which is a coenzyme for a third oxidoreductase following pyridine nucleotide and riboflavin. Research in recent decades has found that PQQ has multiple physiological functions, is a cofactor of many bacterial dehydrogenases, and is involved in the transmission of the electronic respiratory chain of cells; PQQ can improve the stress resistance of microbial cells and improve the oxidation resistance of host bacteria; PQQ is also an animal or plant stimulating or growth factor; PQQ is bound to glucose dehydrogenase to form a PQQ-GDH holoenzyme, and thus, it can be used as a biosensor for detecting glucose and a biofuel cell.
Because the chemical synthesis steps of PQQ are complex and many byproducts are produced at present, biological methods become the most promising industrial production route. PQQ is widely distributed in the biological world, and is found in cells of both animals, plants and microorganisms, but only some gram-negative bacteria can synthesize PQQ. Among the bacteria having the ability to synthesize PQQ, most of them synthesize only trace amounts of PQQ for cell growth, and only a small amount can produce excess PQQ and excrete it into the culture medium, with methylotrophs having the strongest synthesizing ability, such as Paracoccus (Paracoccus), Hyphomicrobium (Hyphomicrobium), Methylobacterium (Methylobacterium), Methylomonas (Methylomonas), Methylophilus (Methylophilus), and Methylobacterium (Methylobacterium), etc. The methylotrophic bacteria are gram-negative bacteria which grow by taking C1 compounds such as methanol and the like as the only carbon source and energy, PQQ is synthesized in cytoplasm along with the growth of the bacteria, and then the PQQ is transported to intercellular substance to combine with methanol dehydrogenase to oxidize methanol so as to provide carbon skeleton for the growth of cells. However, PQQ and methanol dehydrogenase are linked by coordination bonds mediated by Ca2+ or Mg2+, and thus have weak binding force and are easily released to the outside of cells during transport, resulting in the excretion of a large amount of PQQ into the medium.
Although PQQ has been studied for decades, its biological routes vary, the yield of biosynthesis varies greatly, and the overall yield is not high, so that screening strains that produce high PQQ yield from the environment or laboratory is still an indispensable research.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a method for rapidly screening a microorganism capable of producing PQQ, and solves the problem of how to screen a strain with high PQQ yield from the environment or a laboratory.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a method for rapidly screening microorganisms capable of producing PQQ comprises the following steps:
s1, enrichment culture of microbial flora: selecting a microbial flora, inoculating the microbial flora to a methanol enrichment culture solution, and carrying out enrichment culture to obtain an enrichment culture solution;
s2, screening of microbial flora: selecting an enrichment culture solution, coating the enrichment culture solution on a methanol screening flat plate, and continuously increasing the content of methanol to obtain a strain which rapidly grows by using the methanol;
s3, identifying a microbial PQQ synthetic gene cluster: selecting single colonies with different forms from a methanol screening plate, inoculating the single colonies into a fermentation medium, culturing for 1-2 days, quickly extracting a microbial genome, and performing PCR analysis by using characteristic identification primers;
s4, identifying the PQQ produced by the microorganism: transferring the bacterial fluid successfully identified and identified by the PQQ synthetic gene cluster into a fermentation medium, culturing for 3-5 days, centrifuging the fermentation liquor, taking supernatant, rapidly detecting the content of PQQ by a spectrum method, and accurately quantifying the strain with higher yield of PQQ by an HPLC method;
the microbial flora in S1 is a raw filamentous microbe and a paracoccus microbe which are obtained by screening sludge from a methanol production sewage plant by a conventional method;
the screening of the microbial population described in S2 specifically includes the steps of:
(1) selecting an enrichment culture solution, and coating the enrichment culture solution on a screening culture medium plate containing methanol;
(2) testing the content of methanol in the screening medium flat plate, and sequentially increasing the concentration of the methanol from 1% to 8%;
(3) the colony growing under the former methanol concentration is washed out from the plate by sterile water and coated on a screening plate with the next methanol concentration until the screening is finished;
the identification of the microbial PQQ synthetic gene cluster in S3 is carried out by extracting DNA according to a commercialized bacterial genome DNA extraction kit, carrying out PCR by using a known bacterial identification universal primer, amplifying a 16S rDNA sequence of the strain, wherein the success of PCR not only represents the success of genome extraction, but also can identify the species of the strain, carrying out PCR identification by using a characteristic primer designed in a conserved region of the methylotrophic microbial PQQ synthetic gene cluster, and amplifying at least one PQQ synthetase related sequence to prove that the strain has the PQQ synthetic gene cluster, wherein the nucleotide sequence of the characteristic primer is shown in the following sequence table SEQ ID NO. 3-8:
Figure BDA0003077117990000031
Figure BDA0003077117990000041
Figure BDA0003077117990000051
preferably, the composition of the methanol enrichment culture solution in S1 is that 0.1-1%, more preferably 0.5% of methanol is contained in 1L of culture solution, the temperature of enrichment culture is 25-35 ℃, the pH value is 6.0-8.0, and the culture time is 12-72 h.
Preferably, the concentration of methanol in the methanol screening flat plate in S2 is 1% -8%, more preferably 1% -7%, and most preferably 1% -6%.
Preferably, the concentration of methanol in the fermentation culture medium of S3 and S4 is 1-6%, more preferably 2-4%, and most preferably 2.5%; the volume ratio of the switching in the S4 is 1: 1000-1: 5.
Preferably, the methanol-enriched culture solution comprises one or more of ammonium sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, and calcium chloride.
Preferably, the methanol screening plate comprises one or more of ammonium sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, calcium chloride and agar.
Preferably, the fermentation medium comprises one or more of ammonium sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, trace elements and vitamin complex.
Preferably, the trace element composition contains one or more of ferrous sulfate, zinc sulfate, manganese sulfate, copper sulfate, cobalt chloride, boric acid, ammonium molybdate and potassium iodide.
Preferably, the vitamin complex composition contains one or more of riboflavin, pyridoxine, thiamine, calcium pantothenate, nicotinic acid, p-aminobenzoic acid, biotin, folic acid, and inositol.
Preferably, the method can be applied to known PQQ production strains, and a mutant strain with high pyrroloquinoline quinone yield is bred through adaptive domestication on methanol.
(III) advantageous effects
The invention provides a method for rapidly screening microorganisms capable of producing PQQ. The method has the following beneficial effects:
1. according to the herbal plant essence skin-care balance cream and the preparation method thereof, disclosed by the invention, the PQQ high-yield strain is bred by directionally screening or domesticating in a laboratory by taking methanol as an antagonistic factor, so that the problem that the efficiency of screening the PQQ high-yield strain in the traditional environment or laboratory is not high is effectively solved, in addition, the method can also be applied to the known PQQ production strain, and the mutant strain with the high-yield pyrroloquinoline quinone is bred by adaptively domesticating the methanol.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example (b):
the embodiment of the invention provides a method for rapidly screening microorganisms capable of producing PQQ, which comprises the following steps:
s1, enrichment culture of microbial flora: selecting a microbial flora, inoculating the microbial flora to a methanol enrichment culture solution, and carrying out enrichment culture to obtain an enrichment culture solution;
s2, screening of microbial flora: selecting an enrichment culture solution, coating the enrichment culture solution on a methanol screening flat plate, and continuously increasing the content of methanol to obtain a strain which rapidly grows by using the methanol;
s3, identifying a microbial PQQ synthetic gene cluster: selecting single colonies with different forms from a methanol screening plate, inoculating the single colonies into a fermentation medium, culturing for 1-2 days, quickly extracting a microbial genome, and performing PCR analysis by using characteristic identification primers;
s4, identifying the PQQ produced by the microorganism: transferring the bacterial fluid successfully identified and identified by the PQQ synthetic gene cluster into a fermentation medium, culturing for 3-5 days, centrifuging the fermentation liquor, taking supernatant, rapidly detecting the content of PQQ by a spectrum method, and accurately quantifying the strain with higher yield of PQQ by an HPLC method;
the microbial flora in S1 is a raw filamentous microbe and a paracoccus microbe which are obtained by screening sludge from a methanol production sewage plant by a conventional method;
the screening of the microbial population described in S2 specifically includes the steps of:
(1) selecting an enrichment culture solution, and coating the enrichment culture solution on a screening culture medium plate containing methanol;
(2) testing the content of methanol in the screening medium flat plate, and sequentially increasing the concentration of the methanol from 1% to 8%;
(3) the colony growing under the former methanol concentration is washed out from the plate by sterile water and coated on a screening plate with the next methanol concentration until the screening is finished;
the identification of the microorganism PQQ synthetic gene cluster in S3 is to extract DNA according to a commercialized bacterial genome DNA extraction kit, use a known bacterial identification universal primer to carry out PCR, amplify the 16S rDNA sequence of the strain, the success of PCR not only represents the success of genome extraction, but also can identify the species of the strain, use a characteristic primer designed by a conserved region of the methylotrophic microorganism PQQ synthetic gene cluster to carry out PCR identification, and can amplify at least one PQQ synthetase related sequence, thereby proving that the strain has the PQQ synthetic gene cluster.
The methanol enrichment medium formula comprises: (NH)4)2SO43g、KH2PO42g，Na2HPO4·12H2O 3g、MgSO4·7H2O 1g、CaCl2·2H2O 0.5g、FeSO4·7H2O10 mg, pH 6.8, tap water to a constant volume of 1L, adding 5g of sterile methanol after moist heat sterilization, and screening a plate formulation with methanol: (NH)4)2SO43g、KH2PO42g，Na2HPO4·12H2O 3g、MgSO4·7H2O 1g、CaCl2·2H2O 0.5g、FeSO4·7H2O10 mg, agar powder 2g, pH 6.8, constant volume of tap water to 1L, adding 10-80 g of sterile methanol after moist heat sterilization, and the formula of a fermentation medium is as follows: (NH)4)2SO46g、KH2PO44g、Na2HPO4·12H2O 6g、MgSO4·7H2O 1g、CaCl2·2H20.5g of O, 1mL of trace element liquid, 2mL of vitamin auxiliary liquid, pH 6.8, constant volume of tap water to 1L, moist heat sterilization, addition of 25g of sterile methanol and formulation of trace element liquid (1000X): FeSO4·7H2O 5g、CoCl·6H2O 2g、ZnSO4·7H2O 1.4g、MnSO4·H2O 1.6g、CuSO4·5H2O 200mg、(NH4)6Mo7O24·4H2O 40mg、KI 20mg、H3BO360mg, 5mL of 2M HCl, pure water with constant volume to 1L, preservation at 4 ℃, and a formula of vitamin auxiliary liquid (500 x): 0.2g of riboflavin, 0.4g of pyridoxine, 0.4g of thiamine, 0.4g of calcium pantothenate, 0.4g of nicotinic acid, 0.2g of p-aminobenzoic acid, 0.02g of biotin, 0.02g of folic acid and 2g of inositol, wherein the volume of purified water is constant to 1L, and the riboflavin, the biotin and the inositol are stored at 4 ℃.
The bacterial DNA extraction kit is purchased from Tiangen company or a magnetic bead method artificial extraction kit of Biobase company, and the PCR enzyme is replaced by TOYOBO company, and similar products of other companies can also be selected.
Enrichment culture: weighing 0.1g of soil and 50mL of sterile water, adding the soil and the sterile water into a 250mL triangular flask, placing the triangular flask in a shaking table, shaking the triangular flask at the temperature of 28 ℃ and the speed of 200rpm for 2 hours, taking out the triangular flask, and placing the triangular flask at room temperature for standing for 0.5 hour; inoculating 1mL of the supernatant into 50mL of sterile methanol-rich culture solution, placing in a shaking table, and shake-culturing at 28 deg.C and 200rpm for 2 days
Screening methanol: taking 0.1mL of enrichment culture solution, coating the enrichment culture solution on a screening plate containing 1% methanol, culturing for 3 days at 28 ℃, and observing the growth speed of bacterial colonies; washing all colonies on the plate with 1mL of sterile water, taking 0.1mL of the military bacteria liquid, coating the military bacteria liquid on a screening plate containing 2% methanol, culturing at 28 ℃ for 3 days, and observing the growth speed of the colonies; and repeating the steps until colonies grow on a screening plate with 6% methanol, and observing the growth speed of the colonies.
Colonies on the methanol screening plate were picked, inoculated into 5mL of a fermentation medium, and shake-cultured at 28 ℃ and 200rpm for 2 days. 1mL of fermentation broth is taken, and the total genomic DNA of the bacteria is extracted according to the instruction of the kit.
The 16s rDNA sequence of the strain is amplified through PCR reaction by using the bacterial universal primers (SEQ ID No. 1-2), and an obvious PCR product can prove that the genome DNA is successfully extracted. Entrusted Shanghai's work with PCR products to sequence, carried out BLAST comparison on the sequencing result in NCBI website, and identified the species of the strain as: paracoccus and Pediococcus.
Comparing PQQ synthetic gene cluster sequences of methylotrophic microorganisms, analyzing a conserved region, designing characteristic primers (SEQ ID NO. 3-8) according to the conserved region, amplifying the PQQ synthetic gene cluster sequences of the strain through PCR reaction, wherein at least one obvious PCR product can prove that the PQQ synthetic gene cluster is possessed in the genome of the microorganisms. The PCR product can be linked to a T vector by TA cloning, sequencing analysis can be performed by a sequencing company such as Shanghai Biotech, and BLAST comparison can be performed on the sequencing result in NCBI website to confirm that the PQQ synthetic gene is obtained and the strain species can be confirmed.
Colonies on the methanol screening plate were picked, inoculated into 50mL of a fermentation medium, and shake-cultured at 28 ℃ and 200rpm for 5 days. Taking 1mL of fermentation liquor, centrifuging and taking supernatant, finding that the supernatant of the fermentation liquor of part of strains has two absorption peaks at about 249nm and 330nm through full-wavelength scanning, and proving that PQQ exists in the fermentation liquor, and the concentration of the PQQ has a linear relation with OD 330. The two samples with the highest OD 330 readings were taken for liquid phase analysis.
The references are known: the yield of PQQ can be analyzed by using a pure water-resistant COSMOSIL Packed Column 5C18-PAQ reverse phase chromatography Column; the sample amount is 20 mu L, the temperature is 30 ℃, the mobile phase is 0.05mol/L acetic acid, 0.05mol/L ammonium acetate is 30:70, the flow rate is 1mL/min, the content of a PQQ standard substance and a sample is detected under the wavelength of 330nm, and the PQQ peak about 3.56 min; the PQQ concentration and the peak area are in positive correlation; and drawing a standard curve by taking the PQQ concentration as an abscissa and the chromatographic peak area as an ordinate, wherein the linear relation is that y is 30.27x + 7.32.
The liquid phase analysis of the two samples with the highest OD 330 readings shows that: the yield of PQQ in unoptimized shake flasks reached 16.6mg/L and 29.7mg/L, respectively. For the original strain, the PQQ yield belongs to a high-yield line and has important research and application values.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (10)

1. A method for rapidly screening microorganisms capable of producing PQQ is characterized in that: the method specifically comprises the following steps:
s1, enrichment culture of microbial flora: selecting a microbial flora, inoculating the microbial flora to a methanol enrichment culture solution, and carrying out enrichment culture to obtain an enrichment culture solution;
s2, screening of microbial flora: selecting an enrichment culture solution, coating the enrichment culture solution on a methanol screening flat plate, and continuously increasing the content of methanol to obtain a strain which rapidly grows by using the methanol;
s3, identifying a microbial PQQ synthetic gene cluster: selecting single colonies with different forms from a methanol screening plate, inoculating the single colonies into a fermentation medium, culturing for 1-2 days, quickly extracting a microbial genome, and performing PCR analysis by using characteristic identification primers;
s4, identifying the PQQ produced by the microorganism: transferring the bacterial fluid successfully identified and identified by the PQQ synthetic gene cluster into a fermentation medium, culturing for 3-5 days, centrifuging the fermentation liquor, taking supernatant, rapidly detecting the content of PQQ by a spectrum method, and accurately quantifying the strain with higher yield of PQQ by an HPLC method;
the microbial flora in S1 is a raw filamentous microbe and a paracoccus microbe which are obtained by screening sludge from a methanol production sewage plant by a conventional method;
the screening of the microbial population described in S2 specifically includes the steps of:
(1) selecting an enrichment culture solution, and coating the enrichment culture solution on a screening culture medium plate containing methanol;
(2) testing the content of methanol in the screening medium flat plate, and sequentially increasing the concentration of the methanol from 1% to 8%;
(3) the colony growing under the former methanol concentration is washed out from the plate by sterile water and coated on a screening plate with the next methanol concentration until the screening is finished;
the identification of the microorganism PQQ synthetic gene cluster in S3 is to extract DNA according to a commercialized bacterial genome DNA extraction kit, use a known bacterial identification universal primer to carry out PCR, amplify the 16S rDNA sequence of the strain, the success of PCR not only represents the success of genome extraction, but also can identify the species of the strain, use a characteristic primer designed by a conserved region of the methylotrophic microorganism PQQ synthetic gene cluster to carry out PCR identification, and can amplify at least one PQQ synthetase related sequence, thereby proving that the strain has the PQQ synthetic gene cluster.
2. The method of claim 1, which comprises rapidly selecting PQQ-producing microorganisms, wherein the method comprises: the composition of the methanol enrichment culture solution in the S1 is that 1L of culture solution contains 0.1-1%, preferably 0.5%, the temperature of enrichment culture is 25-35 ℃, the pH value is 6.0-8.0, and the culture time is 12-72 h.
3. The method of claim 1, which comprises rapidly selecting PQQ-producing microorganisms, wherein the method comprises: in the S2 methanol screening flat plate, the concentration of methanol is 1-8%, more preferably 1-7%, and most preferably 1-6%.
4. The method of claim 1, which comprises rapidly selecting PQQ-producing microorganisms, wherein the method comprises: the concentration of methanol in the fermentation culture medium of S3 and S4 is 1-6%, more preferably 2-4%, and most preferably 2.5%; the volume ratio of the switching in the S4 is 1: 1000-1: 5.
5. The method of claim 2, which comprises rapidly selecting PQQ-producing microorganisms, wherein the method comprises: the methanol enrichment culture solution contains one or more of ammonium sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate and calcium chloride.
6. The method of claim 3, which comprises rapidly selecting PQQ-producing microorganisms, wherein the method comprises: the methanol screening plate comprises one or more of ammonium sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, calcium chloride and agar.
7. The method of claim 4, wherein the PQQ-producing microorganism is rapidly selected from the group consisting of: the fermentation medium comprises one or more of ammonium sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, trace elements and vitamin complex.
8. The method of claim 7, which comprises rapidly selecting PQQ-producing microorganisms, wherein the method comprises: the trace element composition contains one or more of ferrous sulfate, zinc sulfate, manganese sulfate, copper sulfate, cobalt chloride, boric acid, ammonium molybdate and potassium iodide.
9. The method of claim 7, which comprises rapidly selecting PQQ-producing microorganisms, wherein the method comprises: the vitamin complex composition contains one or more of riboflavin, pyridoxine, thiamine, calcium pantothenate, nicotinic acid, p-aminobenzoic acid, biotin, folic acid, and inositol.
10. The method of claim 1, which comprises rapidly selecting PQQ-producing microorganisms, wherein the method comprises: the method can be applied to known PQQ production strains, and the mutant strains with high pyrroloquinoline quinone yield are bred through adaptive domestication on methanol.
CN202110555665.9A 2021-05-21 2021-05-21 Method for rapidly screening microorganisms capable of producing PQQ Pending CN113308398A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110555665.9A CN113308398A (en) 2021-05-21 2021-05-21 Method for rapidly screening microorganisms capable of producing PQQ

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110555665.9A CN113308398A (en) 2021-05-21 2021-05-21 Method for rapidly screening microorganisms capable of producing PQQ

Publications (1)

Publication Number Publication Date
CN113308398A true CN113308398A (en) 2021-08-27

Family

ID=77374059

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110555665.9A Pending CN113308398A (en) 2021-05-21 2021-05-21 Method for rapidly screening microorganisms capable of producing PQQ

Country Status (1)

Country Link
CN (1) CN113308398A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5344768A (en) * 1985-04-24 1994-09-06 Mitsubishi Gas Chemical Co., Inc. Process for the preparation of pyrrolo-quinoline quinone
WO2012118225A1 (en) * 2011-03-03 2012-09-07 Ajinomoto Co.,Inc. A method for producing pyrroloquinoline quinone using a bacterium of the genus methylobacterium or hyphomicrobium
CN105624084A (en) * 2016-01-28 2016-06-01 福建师范大学 Oriented domestication and breeding of methylotrophic bacterium capable of producing pyrroloquinoline quinone at high yield

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5344768A (en) * 1985-04-24 1994-09-06 Mitsubishi Gas Chemical Co., Inc. Process for the preparation of pyrrolo-quinoline quinone
WO2012118225A1 (en) * 2011-03-03 2012-09-07 Ajinomoto Co.,Inc. A method for producing pyrroloquinoline quinone using a bacterium of the genus methylobacterium or hyphomicrobium
CN105624084A (en) * 2016-01-28 2016-06-01 福建师范大学 Oriented domestication and breeding of methylotrophic bacterium capable of producing pyrroloquinoline quinone at high yield

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
徐文等: "甲醇利用型吡咯喹啉醌产生菌的筛选及鉴定", 《生物技术通报》 *
杨亚欣等: "一株吡咯喹啉醌产生菌的筛选和初步鉴定", 《生物技术通讯》 *
柯崇榕: "适应性驯化选育高产吡咯喹啉醌的生丝微菌突变株", 《生物工程学报》 *
王歆等: "吡咯喹啉醌产生菌筛选方法建立及菌种筛选", 《微生物学报》 *

Similar Documents

Publication Publication Date Title
Yu et al. Syntrophic pathways for microbial mercury methylation
Myhr et al. Denitrovibrio acetiphilus, a novel genus and species of dissimilatory nitrate-reducing bacterium isolated from an oil reservoir model column.
Pattanamanee et al. Photofermentive production of biohydrogen from oil palm waste hydrolysate
Fujimura et al. Analysis of early bacterial communities on volcanic deposits on the island of Miyake (Miyake-jima), Japan: a 6-year study at a fixed site
JPH08116964A (en) Biomass containing l-ascorbic acid
Peschek Structure and function of respiratory membranes in cyanobacteria (blue-green algae)
Si et al. Novel and efficient screening of PQQ high-yielding strains and subsequent cultivation optimization
CN102559832B (en) Method for com-culturing and improving hydrogen output by utilizing bacteria and chlamydomonas reinhardtii
CN114854630A (en) Selenium-resistant bacillus and breeding method and application thereof
Bagwell et al. A diverse assemblage of indole-3-acetic acid producing bacteria associate with unicellular green algae
CN113373072B (en) Heterotrophic nitrification aerobic denitrification fungus strain and separation method and application thereof
CN104630100A (en) Reconstructed Klebsiella pneumoniae and application of reconstructed Klebsiella pneumoniae in production of R-acetoin
Tornabene Non-aerated cultivation of Halobacterium cutirubrum and its effect on cellular squalenes
CN113308398A (en) Method for rapidly screening microorganisms capable of producing PQQ
Aung et al. Isolation of endophytic bacteria from green gram and study on their plant growth promoting activities
Silva et al. Effect of mineral nutrients on cell growth and self-flocculation of Tolypothrix tenuis for the production of a biofertilizer
CN112063574B (en) Method for improving lycopene yield of Blakeslea trispora
CN105670965B (en) Strain with iron reduction capacity and application thereof
CN111534534A (en) Method for producing high-selenium-content protein by constructing yeast fermentation by using exogenous metallothionein
Ladha et al. The soil microbiome and crop nitrogen nutrition i n anaerobic system: a case study in rice
CN116103178B (en) Copper-resistant pichia pastoris strain with high copper enrichment and application thereof
CN113215064B (en) Slime bacterium for producing meishadazole compounds and application thereof
Colsell Cultivation, isolation and characterisation of thermophilic and acidophilic red algae, Cyanidiales.
CN117946914A (en) High-yield rhamnolipid strain, microbial inoculum, method for producing rhamnolipid and application
Liao et al. Isolation and identification of selenium-tolerant strains from selenium-rich soil in Guangxi

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20210827