CN107488619A - The monad SXZ N10 bacterial strains and purposes of one plant of production huperzine and Huperzine B - Google Patents

The monad SXZ N10 bacterial strains and purposes of one plant of production huperzine and Huperzine B Download PDF

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CN107488619A
CN107488619A CN201710923780.0A CN201710923780A CN107488619A CN 107488619 A CN107488619 A CN 107488619A CN 201710923780 A CN201710923780 A CN 201710923780A CN 107488619 A CN107488619 A CN 107488619A
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huperzine
sxz
monad
bacterial strain
fermentation
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CN107488619B (en
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张鹏
张学智
宋发军
刘盼
裴婷
吴新广
耿红
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South Central Minzu University
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    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system

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Abstract

The invention provides one plant of production huperzine and the monad SXZ N10 bacterial strains of Huperzine B, the bacterial strain can produce huperzine and Huperzine B.The bacterial strain is named as SXZ N10, belongs to a kind of monad, and the bacterial strain is preserved in China typical culture collection center, address：China, Wuhan, Wuhan University；Postcode 430072, preservation date are September in 2017 14, and deposit number is：CCTCC NO：M2017512；The 16S rDNA sequences of the bacterial strain are as shown in SEQ ID NO.1.Above-mentioned monad SXZ N10 bacterial strains can be used in fermentation and prepare huperzine and Huperzine B.Condition of microbe fermentation includes：Fermentation medium forms：Dusty yeast 8g/L, peptone 5g/L, sodium chloride 5g/L, glucose 10g/L, pH 6.0；Condition of culture is：27 DEG C, 200rpm, fermented and cultured 7 days.

Description

The monad SXZ-N10 bacterial strains and purposes of one plant of production huperzine and Huperzine B
Technical field
The present invention relates to a kind of aeromonas strain, the bacterial strain can produce huperzine and Huperzine B, belong to microbial technique Field.
Background technology
Endophyte of plant research at present is gradually taken seriously, and the various endophytes of different plants are found in succession.Endophyte The diversity of species also gives the diversity of its metabolite, this also imply that using microbial metabolism excavate biology, chemistry, The new resources such as medicine have huge potentiality.Numerous studies show that endophyte of plant can produce same or similar with host plant Active material and precursor, this discovery pushed directly on endophyte of plant especially medicinal plant endophyte research extensive exhibition Open.With progressively research of the people to medicinal plant endophyte, using can be metabolized the endophyte that produces corresponding active component come New, efficient, inexpensive active medicine is produced, the present situation for solving rare Chinese medicinal plant resource scarcity, medicine in future will be turned into The emphasis studied with endophyte of plant.
Medicinal active ingredient huperzine (Huperzine A, HupA) and Huperzine B in Huperzia serrata (Huperzine B, HupB) is a kind of less toxic, efficient acetylcholine esterase inhibition (Acetyl cholinesterase, AChE) Medicine, wherein huperzine be clinically be used for treat " Alzheimer's disease (senile dementia) " (Alzaheimer Disease, AD) key agents.However, due to Huperzia serrata plant resources is deficient and slow-growing, huperzine content very Low reason, the market demand can not be met by only relying on from Huperzia serrata extraction huperzine.And the huperzine of chemical synthesis Activity is far below natural huperzine, and the huperzine cost for obtaining high optical activity is higher.Have in the art Microbial fermentation is carried out so as to produce the relevant report of huperzine using Huperzia serrata endogenetic bacterium, but the species of these bacterial strains And yield still can not meet the needs of market is for huperzine.
The content of the invention
The present invention is solved the problems, such as in background technology, there is provided the monad of one plant of production huperzine and Huperzine B SXZ-N10 bacterial strains, the bacterial strain can produce huperzine and Huperzine B.
The present inventor screens one plant of novel strain, and the bacterial strain is named as SXZ-N10, belongs to a kind of monad (Brevundimonas sp.), the bacterial strain is preserved in China typical culture collection center, address：China, Wuhan, Wuhan are big Learn；Postcode 430072, preservation date are September in 2017 14, and deposit number is：CCTCC NO： M2017512；The bacterial strain 16S rDNA sequences are as shown in SEQ ID NO.1.
Above-mentioned monad SXZ-N10 bacterial strains can be used in fermentation and prepare huperzine and Huperzine B.Microbial fermentation bar Part includes：Fermentation medium forms：Dusty yeast 8g/L, peptone 5g/L, sodium chloride 5g/L, glucose 10g/L, pH 6.0；Training Foster condition is：27 DEG C, 200rpm, fermented and cultured 7 days.
Compared with prior art, the present invention has advantages below：Monad SXZ-N10 bacterial strains provided by the present invention can Metabolism produces huperzine and Huperzine B.Bacterial strain provided by the invention can carry out large scale fermentation culture in a short time, and Fermentation costs are relatively low, and are not limited by conditions such as time domain, seasons, therefore can solve huperzine by the approach of microbial fermentation The present situation that first cost is higher, market demand breach is big.Simultaneously the present invention in additionally provide optimization after condition of microbe fermentation with And the formula of culture medium, the yield of huperzine and Huperzine B are higher.
Brief description of the drawings
Fig. 1 is the huperzine of monad SXZ-N10 bacterial strains and the first time HPLC detection figures of Huperzine B；
Fig. 2 is the huperzine of monad SXZ-N10 bacterial strains and second of HPLC detection figures of Huperzine B；
Fig. 3 is the MS detection figures of the huperzine of monad SXZ-N10 bacterial strains；
Fig. 4 is the MS detection figures of the Huperzine B of monad SXZ-N10 bacterial strains.
Embodiment
Detailed specific description is done to the present invention with reference to specific embodiment, but protection scope of the present invention not office It is limited to following examples.
Separation, the culture of bacterial strain
After Huperzia serrata material is rinsed well with clear water in the present embodiment, then with clear water ultrasound 10min, separate its leaf, Stem simultaneously cuts into about 4cm segments, is soaked 2 minutes in 0.1% mercuric chloride soaks 4min, 70% ethanol successively, the leaching of 5% sodium hypochlorite Bubble 5 minutes, is repeatedly rinsed with sterilized water, homogenate is ground under aseptic condition, put down in each PDA without antibiotic afterwards 200 μ l homogenate coatings, 30 DEG C of quiescent culture 1-2d are taken on plate.According to bacterium at the random picking 20 of bacterial growth situation in incubation Fall, be connected to new LA culture mediums, and each monoclonal, numbering and conservation are obtained by the separation passage of multiple plate streak.
By the bacterium picking that completion is purified on flat board into the 1.5mL EP pipes equipped with 600 μ L LB fluid nutrient mediums, seal Film seals, and then in 37 DEG C of constant-temperature tables after 190r/min shaking table cultures 1d, 50% isometric glycerine is added, after mixing Preserved in -80 DEG C of ultra low temperature freezers, each bacterial strain at least preserves 3 parts.
Take 10 μ L to be inoculated into 5mL LB fluid nutrient mediums the strain of preservation, lived in 37 DEG C of constant-temperature table 150r/min Change culture, take in the LB fluid nutrient mediums in strain to the triangular flask of 3mL activation and fermented after 24h, liquid amount 60mL/ 100mL, 37 DEG C, 150r/min shaking table cultures 7d.After above fermented and cultured, zymotic fluid is collected.The screening of bacterial strain
The bacterial strain that above-mentioned collection obtains is detected by high performance liquid chromatography (HPLC) primary dcreening operation and secondary screening in the present embodiment After the extractive from fermentative of each endogenetic bacteria, further confirmed that using mass spectrography (Mass Spectrometry, MS).Separate The bacterial strain that can produce huperzine and Huperzine B to one plant, is named as：SXZ-N10 bacterial strains.
HPLC primary dcreening operations：Chromatograph is ultimate-3000 chromatographic systems, ODS-C18 reversed-phase columns (4.6mm × 150mm, 5 μ M, Agilent), mobile phase is methanol：Water=65:35；Standard concentration is 100 μ g/mL, flow velocity 0.5mL/min, ultraviolet waves Long to be set as 310nm, column temperature is 20 DEG C, and testing result is as shown in Figure 1.
HPLC secondary screenings：Change mobile phase is methanol：Water=55:45, other conditions are constant.Testing result is as shown in Figure 2.
Mass spectral analysis uses liquid chromatography-mass spectrometry at South-Center University For Nationalities's Hua Cai institutes test analysis center Instrument/systems of Agilent LC-Q-TOF-MS 6520 are completed.The wherein MS detections of the huperzine of monad SXZ-N10 bacterial strains As shown in Figure 3；The MS detections of the Huperzine B of monad SXZ-N10 bacterial strains are as shown in Figure 4.
Sequencing and sequence alignment, analysis
Certain Bioisystech Co., Ltd is given to be sequenced the bacterial strain, the 16S rDNA sequences such as SEQ ID NO.1 institutes of the bacterial strain Show.Sequencing result carries out Blast similarity analysis in GenBank nucleic acid databases, through Blast sequence alignments and chadogram point Analysis, determine that it is monad (Brevundimonas sp.) bacterium.
The preservation of bacterial strain：
The bacterial strain is preserved in China typical culture collection center, address：China, Wuhan, Wuhan University；Postcode 430072, preservation date is September in 2017 14, and deposit number is：CCTCC NO：M2017512.
The optimization of fermentation condition
The fermentation medium component of microorganism allows for the needs for meeting the breeding of bacterial strain fast-growth, while most suitable hair Ferment condition of culture and can enough ensures a large amount of accumulation of its secondary metabolites.The preliminary fermentation for optimizing SXZ-N10 bacterial strains of the invention Medium component and condition of culture, the culture medium prescription after optimization are：Dusty yeast 8g/L, peptone 5g/L, sodium chloride 5g/L, Glucose 10g/L, initial pH value 6.0；Condition of culture is：27 DEG C, shaking speed 200r/min of cultivation temperature, fermentation time 7 My god.
Sequence table
<110>South-Center University For Nationalities
<120>The monad SXZ-N10 bacterial strains and purposes of one plant of production huperzine and Huperzine B
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1425
<212> DNA
<213>Monad (Brevundimonas sp.)
<400> 1
agagtttgat catggctcag agcgaacgct ggcggcaggc ctaacacatg caagtcgaac 60
ggacccttcg gggttagtgg cggacgggtg agtaacacgt gggaacgtgc ctttaggttc 120
ggaatagctc ctggaaacgg gtggtaatgc cgaatgtgcc cttcggggga aagatttatc 180
gcctttagag cggcccgcgt ctgattagct agttggtgag gtaatggctc accaaggcga 240
cgatcagtag ctggtctgag aggatgacca gccacattgg gactgagaca cggcccaaac 300
tcctacggga ggcagcagtg gggaatcttg cgcaatgggc gaaagcctga cgcagccatg 360
ccgcgtgaat gatgaaggtc ttaggattgt aaaattcttt caccggggac gataatgacg 420
gtacccggag aagaagcccc ggctaacttc gtgccagcag ccgcggtaat acgaaggggg 480
ctagcgttgc tcggaattac tgggcgtaaa gggcgcgtag gcggacattt aagtcagggg 540
tgaaatccca gagctcaact ctggaactgc ctttgatact gggtgtcttg agtgtgagag 600
aggtatgtgg aactccgagt gtagaggtga aattcgtaga tattcggaag aacaccagtg 660
gcgaaggcga catactggct cattactgac gctgaggcgc gaaagcgtgg ggagcaaaca 720
ggattagata ccctggtagt ccacgccgta aacgatgatt gctagttgtc gggctgcatg 780
cagttcggtg acgcagctaa cgcattaagc aatccgcctg gggagtacgg tcgcaagatt 840
aaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa 900
gcaacgcgca gaaccttacc accttttgac atgcctggac cgccagagag atctggcttt 960
ctcttcggag actaggacac aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt 1020
tgggttaagt cccgcaacga gcgcaaccct cgccattagt tgccatcatt tagttgggaa 1080
ctctaatggg actgccggtg ctaagccgga ggaaggtggg gatgacgtca agtcctcatg 1140
gcccttacag ggtgggctac acacgtgcta caatggcgac tacagagggt taatccttaa 1200
aagtcgtctc agttcggatt gtcctctgca actcgagggc atgaagttgg aatcgctagt 1260
aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1320
caccatggga gttggttcta cccgaaggcg atgcgctaac cgcaaggagg cagtcgacca 1380
cggtagggtc agcgactggg gtgaagtcgt aacaaggtaa ccgta 1425

Claims (3)

1. monad (Brevundimonas sp.) SXZ-N10 bacterial strains of one plant of production huperzine and Huperzine B, the bacterial strain are protected It is hidden in China typical culture collection center, address：China, Wuhan, Wuhan University；Postcode 430072, preservation date 2017 On September 14, deposit number are：CCTCC NO：M2017512, the 16S rDNA sequences of the bacterial strain are as shown in SEQ ID NO.1.
2. the purposes of monad SXZ-N10 bacterial strains described in claim 1, it is characterised in that：For fermentation prepare huperzine and Huperzine B.
3. purposes according to claim 2, it is characterised in that：Condition of microbe fermentation is：Fermentation medium forms：Yeast Powder 8g/L, peptone 5g/L, sodium chloride 5g/L, glucose 10g/L, pH 6.0；Condition of culture is：27 DEG C, 200r/min, fermentation Culture 7 days.
CN201710923780.0A 2017-09-30 2017-09-30 Brevundimonas SXZ-N10 strain for producing huperzine A and huperzine B and application thereof Active CN107488619B (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109749965A (en) * 2019-02-27 2019-05-14 中南民族大学 The bacillus subtilis QNX-7HL bacterial strain and purposes of one plant of production huperzine
CN110468055A (en) * 2019-07-29 2019-11-19 西北大学 Colletotrichum gloeosporioides Penz and its application in a kind of serrate clubmoss herb
CN113373098A (en) * 2021-07-28 2021-09-10 西安医学院 Stenotrophomonas H2 strain for treating Alzheimer disease and application thereof
CN113462594A (en) * 2021-06-17 2021-10-01 中南民族大学 Kluyveromyces QNX-S26 strain for producing huperzine B and application thereof
CN113528391A (en) * 2021-07-28 2021-10-22 西安医学院 Stenotrophomonas H1 strain for efficiently expressing huperzine A and application thereof

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CN101942393A (en) * 2009-12-30 2011-01-12 朱笃 Huperzia serrata endophytic fungus shiraia sp. strain for producing huperzine A
WO2013040206A1 (en) * 2011-09-14 2013-03-21 Lewis Thomas J Novel formulations comprising macrolide and tetracycline and their uses

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CN101942393A (en) * 2009-12-30 2011-01-12 朱笃 Huperzia serrata endophytic fungus shiraia sp. strain for producing huperzine A
WO2013040206A1 (en) * 2011-09-14 2013-03-21 Lewis Thomas J Novel formulations comprising macrolide and tetracycline and their uses

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Title
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孙红敏等: "蛇足石杉内生细菌多样性", 《微生物学报》 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109749965A (en) * 2019-02-27 2019-05-14 中南民族大学 The bacillus subtilis QNX-7HL bacterial strain and purposes of one plant of production huperzine
CN110468055A (en) * 2019-07-29 2019-11-19 西北大学 Colletotrichum gloeosporioides Penz and its application in a kind of serrate clubmoss herb
CN110468055B (en) * 2019-07-29 2021-09-14 西北大学 Huperzia serrata colletotrichum and application thereof
CN113462594A (en) * 2021-06-17 2021-10-01 中南民族大学 Kluyveromyces QNX-S26 strain for producing huperzine B and application thereof
CN113462594B (en) * 2021-06-17 2022-04-15 中南民族大学 Kluyveromyces QNX-S26 strain for producing huperzine B and application thereof
CN113373098A (en) * 2021-07-28 2021-09-10 西安医学院 Stenotrophomonas H2 strain for treating Alzheimer disease and application thereof
CN113528391A (en) * 2021-07-28 2021-10-22 西安医学院 Stenotrophomonas H1 strain for efficiently expressing huperzine A and application thereof
CN113373098B (en) * 2021-07-28 2022-07-01 西安医学院 Stenotrophomonas H2 strain for treating Alzheimer disease and application thereof
CN113528391B (en) * 2021-07-28 2024-05-17 西北大学 Stenotrophomonas H1 strain for efficiently expressing huperzine A and application thereof

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