CN103408550B - Derive from 2,5-diketopiperazines dipeptides and the Synthesis and applications thereof producing the molten bacillus of enzyme - Google Patents
Derive from 2,5-diketopiperazines dipeptides and the Synthesis and applications thereof producing the molten bacillus of enzyme Download PDFInfo
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 33
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 33
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 33
- 108010016626 Dipeptides Proteins 0.000 title claims abstract description 31
- BXRNXXXXHLBUKK-UHFFFAOYSA-N piperazine-2,5-dione Chemical class O=C1CNC(=O)CN1 BXRNXXXXHLBUKK-UHFFFAOYSA-N 0.000 title claims abstract description 26
- 230000015572 biosynthetic process Effects 0.000 title description 2
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- 238000002360 preparation method Methods 0.000 claims abstract description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 18
- 239000000284 extract Substances 0.000 claims description 18
- 241000863030 Lysobacter enzymogenes Species 0.000 claims description 14
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
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- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 2
- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 description 2
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a kind of 2,5-diketopiperazines dipeptides (I) deriving from the molten bacillus of product enzyme, and preparation and application, and produce the endogenetic bacteria of described 2,5-diketopiperazines dipeptides---produce the molten bacillus (Lysobacter of enzyme? enzymogenes) R-2-1.Beneficial effect of the present invention is mainly reflected in: (1) the invention provides a kind of molten bacillus R-2-1 of product enzyme producing 2,5-diketopiperazines dipeptides (I), and this bacterium produces the molten bacillus of enzyme in the first strain; (2) 2,5-diketopiperazines dipeptides (I) of the present invention have very strong restraining effect to flavus and Candida albicans, can be used as new type bactericide, can be used for the medicine preparing treatment flavus or the microbial relative disease of Candida albicans further; (3) 2,5-diketopiperazines dipeptides (I) of the present invention derive from and produce the production of enzyme molten bacillus R-2-1 liquid fermenting, and operating procedure is easy, and the cycle is short, and cost is low, originates guaranteed; (4) the present invention utilizes biological process to synthesize, environmentally safe.
Description
(1) technical field
The present invention relates to one derive from produce the molten bacillus of enzyme (Lysobacter enzymogenes) 2,5-diketopiperazines dipeptides, and preparation and application, and produce the endogenetic bacteria of described 2,5-diketopiperazines dipeptides---produce enzyme molten bacillus (Lysobacter enzymogenes) R-2-1.
(2) background technology
Pathogenic bacterium not only serious harm the health of human body, and threaten agriculture production, bring very large loss to people of other countries' economy and life.Although the existing sterilant huge number of China, novel pathogenic bacterium and strong resistance pathogenic bacterium effectively can not be suppressed.Therefore, sterilant must be constantly brought forth new ideas.Bio-pharmaceutical has efficiently, low toxicity, safety, advantages of environment protection, has catered to the demand of China's drug development, and following microbial medicine industry has very large development potentiality.
Expanding Microbial resources is one of important channels of development of new sterilant.Endophyte of plant is the special microorganism of a class, and it lives between health plant histocyte or in tissue, not causing any obvious Disease symptoms of host plant, is endophytic normal microflora.Large quantity research shows, endophyte of plant distribution is wide, and kind is many, has important physiology and Ecology Action, and endophyte of plant has abundant Chemical Diversity, can unique, the active significant material of metabolic chemistry structure.Up to the present, there is not yet the report being separated to the endophyte product molten bacillus of enzyme (Lysobacter enzymogenes) and metabolism 2,5-diketopiperazines dipeptides thereof from Herba Artemisiae annuae.
(3) summary of the invention
The object of the invention is to provide a kind of 2,5-diketopiperazines dipeptides and preparation and application thereof, and produces the endogenetic bacteria of described 2,5-diketopiperazines dipeptides---produce enzyme molten bacillus (Lysobacterenzymogenes) R-2-1.
The technical solution used in the present invention is:
One derive from produce the molten bacillus of enzyme (Lysobacter enzymogenes) 2,5-diketopiperazines dipeptides, its structure as shown in the formula (I):
Described endogenetic bacteria produces enzyme molten bacillus R-2-1 separation method: fetch fresh and healthy Herba Artemisiae annuae stem (the Artemisia annua Linn. coming from Tianmu Mountains of Zhejiang Province, composite family, artemisia, Herba Artemisiae annuae kind), load sealing mark in freshness protection package, in 24h, carry out endophyte separation.Herba Artemisiae annuae root clear water is cleaned, and is soaked in volumetric concentration 75% aqueous ethanolic solution 1min, mass concentration 1% aqueous sodium hypochlorite solution 10min successively, volumetric concentration 75% aqueous ethanolic solution 1min carries out pre-treatment after being cut into the segment of about 1cm.Get pretreated Herba Artemisiae annuae root to be cultured to bacterium colony with dual anti-WA culture medium flat plate and to grow from incision, picking colony is forwarded to the separation that LB substratum carries out endogenetic fungus, through colonial morphology observe and 16S rRNA Molecular Identification for producing the molten bacillus of enzyme (Lysobacter enzymogenes), called after endogenetic bacteria product enzyme molten bacillus R-2-1(Lysobacter enzymogenes).Dual anti-WA substratum final concentration composition: 200IU/mL amphotericin B, 150IU/mL Streptomycin sulphate, 20g agar, 1L distilled water, natural ph.LB substratum final concentration forms: yeast extract 5g/L, Tryptones 10g/L, NaCl10g/L, agar 20g/L, solvent is water, pH7.4.
Endogenetic bacteria produces enzyme molten bacillus R-2-1 morphological specificity and cultural characteristic is: bacterium colony is light yellow, G
-, cell is shaft-like, and spore staining is purple, and methyl red test is positive, and NaCl content range 0 ~ 3%, has oxidase activity and hydrolyzed casein, carboxy methyl cellulose activity, (G+C) content 66.9% in DNA, nitrate-free reducing power.
This bacterium 16S rRNA sequence is:
1tgcagtcgaa cggcagcaca gaggagcttg ctccttgggt ggcgagtggc ggacgggtga
61ggaatacgtc ggaatctgcc tatttgtggg ggataacgta gggaaactta cgctaatacc
121gcatacgacc tacgggtgaa agtgggggac cgcaaggcct cacgcagata gatgagccga
181cgtcggatta gctagttggc ggggtaaagg cccaccaagg cgacgatccg tagctggtct
241gagaggatga tcagccacac tggaactgag acacggtcca gactcctacg ggaggcagca
301gtggggaata ttggacaatg ggcgcaagcc tgatccagcc atgccgcgtg tgtgaagaag
361gccttcgggt tgtaaagcac ttttgtccgg aaagaaaagc ttagggttaa taaccttgag
421tcatgacggt accggaagaa taagcaccgg ctaacttcgt gccagcagcc gcggtaatac
481gaagggtgca agcgttactc ggaattactg ggcgtaaagc gtgcgtaggt ggtttgttaa
541gtctgatgtg aaagccctgg gctcaacctg ggaatggcat tggaaactgg cttactagag
601tgcggtagag ggtagcggaa ttcccggtgt agcagtgaaa tgcgtagata tcgggaggaa
661catctgtggc gaaggcggct acctggacca gcactgacac tgaggcacga aagcgtgggg
721agcaaacagg attagatacc ctggtagtcc acgccctaaa cgatgcgaac tggatgttgg
781gggcaacttg gccctcagta tcgaagctaa cgcgttaagt tcgccgcctg ggaagtacgg
841tcgcaagact gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agtatgtggt
901ttaattcgat gcaacgcgaa gaaccttacc tggccttgac atgtcgagaa ctttccagag
961atggattggt gccttcggga actcgaacac aggtgctgca tggctgtcgt cagctcgtgt
1021cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgtccttagt tgccagcacg
1081taatggtggg aactctaagg agaccgccgg tgacaaaccg gaggaaggtg gggatgacgt
1141caagtcatca tggcccttac ggccagggct acacacgtac tacaatggta gggacagagg
1201gctgcaaacc cgcgagggca agccaatccc agaaacccta tctcagtccg gattggagtc
1261tgcaactcga ctccatgaag tcggaatcgc tagtaatcgc agatcagcat tgctgcggtg
1321aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgggagtttg ttgcaccaga
1381agcaggtagc ttaaccttcg ggagggcgct gc。
The invention still further relates to described 2, the preparation method of 5-diketopiperazines dipeptides, described method comprises: will produce the filtering fermentation liquor or centrifugal that the molten bacillus of enzyme (Lysobacter enzymogenes) CCTCC NO:M2013203 obtains through fermentation, get filtrate or supernatant liquor is extracted with ethyl acetate, get organic layer to concentrate, gained medicinal extract, through separation and purification, obtains described 2,5-diketopiperazines dipeptides.
Preferably, described separation purification method is as follows: gained medicinal extract is carried out silica gel column chromatography, with chloroform by (1): the solvent of methyl alcohol volume ratio=100:4 is that eluent carries out wash-out, collects elutriant, concentrated, obtains medicinal extract 1; (2) medicinal extract 1 is carried out silica gel column chromatography, with chloroform: the solvent of methyl alcohol volume ratio=100:2 is that eluent carries out wash-out, collect elutriant, concentrated, obtain medicinal extract 2; (3) dextrane gel Sephadex LH-20 is utilized to carry out column chromatography to medicinal extract 2, with chloroform: the solvent of methyl alcohol volume ratio=1:1 is that eluent carries out wash-out, TLC tracing detection contains the part of blue-fluorescence point, collect this elution fractions, after merging elutriant, cryogenic vacuum removes eluent, obtain 2,5-diketopiperazines dipeptides shown in formula (I).
The fermented liquid of the molten bacillus of described product enzyme to be applicable in routine by described product enzyme molten bacillus CCTCC NO:M2013203 to produce in the fermention medium of the molten bacillus of enzyme and to obtain through conventional fermentation culture, usually also needs to cultivate and seed enlarged culturing through slant activation before fermentation culture.
Concrete, described fermented liquid preparation method is as follows:
(1) slant culture: molten for product enzyme bacillus CCTCC NO:M2013203 is seeded to slant medium, cultivates 1 ~ 3 day for 37 DEG C, obtains thalline inclined-plane; Described slant medium final concentration consists of: yeast extract 5g/L, Tryptones 10g/L, NaCl10g/L, agar 20g/L, and solvent is water, pH7.4;
(2) seed culture: be seeded to LB seed culture medium from thalline inclined-plane picking one transfering loop thalline, under 100 ~ 200rpm, 37 DEG C of conditions, shaking table is cultivated 1 ~ 3 day, obtains seed liquor; Described LB seed culture medium final concentration consists of: yeast extract 5g/L, Tryptones 10g/L, NaCl10g/L, and solvent is water, pH7.4;
(3) fermentation culture: the seed liquor that step (2) obtains is seeded to fermention medium with the inoculum size of volume ratio 1:20, under 100 ~ 200rpm, 30 ~ 37 DEG C of conditions, shaking table is cultivated 3 ~ 6 days, obtain fermentation culture, fermention medium final concentration composition is with LB seed culture medium.
The present invention obtain 2,5-diketopiperazines dipeptides (I) has very strong restraining effect to flavus (Aspergillus flavus) and Candida albicans (Candida albicans), so this dipeptides can be used as the compound with bacteriostatic action, be expected to be applied preparing in sterilant.
The invention still further relates to described 2,5-diketopiperazines dipeptides and prepare the application in sterilant.
Preferably, described sterilant is the sterilant suppressing flavus (Aspergillus flavus) or Candida albicans (Candida albicans).Further, in described sterilant, 2,5-diketopiperazines dipeptides effective concentration are 30ug/mL.
Because flavus or Candida albicans are pathogenic bacterium, therefore the compounds of this invention can be used for the medicine preparing treatment flavus or the microbial relative disease of Candida albicans further.
Concrete, the invention still further relates to the application of described 2,5-diketopiperazines dipeptides in the medicine of preparation treatment Candida albicans microbial dermatocandidiasis, candidiasis of the mucous membranes (as white mouth, bridou, vaginitis etc.) or internal organ and nervus centralis moniliosis (as pneumonia, gastroenteritis, endocarditis, meningitis, encephalitis etc.).
The invention still further relates to the application of described 2,5-diketopiperazines dipeptides in the medicine preparing the food poisoning (hepar damnification caused of especially poisoning by food) that treatment flavus causes.
The invention still further relates to and produce described 2, the endogenetic bacteria of 5-diketopiperazines dipeptides---produce enzyme molten bacillus (Lysobacter enzymogenes) R-2-1, be preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, preservation date is on May 14th, 2013, and deposit number is CCTCC NO:M2013203.
Beneficial effect of the present invention is mainly reflected in:
(1) the invention provides a kind of molten bacillus R-2-1 of product enzyme producing 2,5-diketopiperazines dipeptides (I), this bacterium produces the molten bacillus of enzyme in the first strain;
(2) of the present invention 2,5-diketopiperazines dipeptides (I) has very strong restraining effect to flavus (Aspergillus flavus) and Candida albicans (Candida albicans), can be used as new type bactericide, can be used for the medicine preparing treatment flavus or the microbial relative disease of Candida albicans further;
(3) 2,5-diketopiperazines dipeptides (I) of the present invention derive from and produce the production of enzyme molten bacillus R-2-1 liquid fermenting, and operating procedure is easy, and the cycle is short, and cost is low, originates guaranteed;
(4) the present invention utilizes biological process to synthesize, environmentally safe.
(4) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: endophyte produces the isolation and purification of enzyme molten bacillus (Lysobacter enzymogenes) R-2-1
Dual anti-WA substratum final concentration composition: 200IU/mL amphotericin B, 150IU/mL Streptomycin sulphate, 20g agar, 1L distilled water, natural ph.
LB substratum final concentration forms: yeast extract 5g/L, Tryptones 10g/L, NaCl10g/L, agar 20g/L, solvent is water, pH7.4.
Get the Herba Artemisiae annuae root (Artemisia annua Linn., composite family, artemisia, Herba Artemisiae annuae kind) of fresh and healthy, load sealing mark in freshness protection package, in 24h, carry out endophyte separation.Herba Artemisiae annuae root clear water is cleaned, and is soaked in volumetric concentration 75% aqueous ethanolic solution 1min, mass concentration 1% aqueous sodium hypochlorite solution 10min successively, volumetric concentration 75% aqueous ethanolic solution 1min carries out pre-treatment after being cut into the segment of about 1cm.Get after the dual anti-WA culture medium flat plate of pretreated Herba Artemisiae annuae root cultivates 2 ~ 4 days at 28 ~ 37 DEG C, bacterium colony grows from incision, picking colony is forwarded to LB substratum and is separated, through colonial morphology observe and 16S rRNA Molecular Identification for product the molten bacillus of enzyme (Lysobacter enzymogenes), called after endogenetic bacteria produces enzyme molten bacillus (Lysobacterenzymogenes) R-2-1, this bacterial strain is preserved in China typical culture collection center, preservation date on May 14th, 2013, deposit number is CCTCC NO:M2013203.
It is light yellow that endogenetic bacteria produces enzyme molten bacillus R-2-1 bacterium colony, G-, and cell is shaft-like, spore staining is purple, and methyl red test is positive, NaCl content range 0 ~ 3%, there is oxidase activity and hydrolyzed casein, carboxy methyl cellulose activity, (G+C) content 66.9% in DNA, nitrate-free reducing power.
This bacterium 16S rRNA sequence is see SEQ ID No.1.
Embodiment 2: endophyte produces the preparation of enzyme molten bacillus R-2-1 fermentation culture
(1) slant culture: endogenetic fungus is produced the molten bacillus R-2-1 of enzyme and be seeded to slant medium, cultivates 2 days for 37 DEG C, obtains thalline inclined-plane; Described slant medium final concentration consists of: yeast extract 5g/L, Tryptones 10g/L, NaCl10g/L, agar 20g/L, and solvent is water, pH7.4;
(2) seed culture: be seeded to LB seed culture medium from thalline inclined-plane picking one transfering loop thalline, shaking table cultivates 2 days under 100 ~ 200rpm, 37 DEG C of conditions, obtains seed liquor; Described LB seed culture medium final concentration consists of: yeast extract 5g/L, Tryptones 10g/L, NaCl10g/L, and solvent is water, pH7.4;
(3) fermentation culture: the seed liquor that step (2) obtains is seeded to fermention medium with the inoculum size of volume ratio 1:20, under 100 ~ 200rpm, 30 ~ 37 DEG C of conditions, shaking table is cultivated 3 ~ 6 days, obtain fermentation culture, fermention medium final concentration composition is with LB seed culture medium.
Embodiment 3: alkaloidal Extraction and isolation, qualification
1, alkaloidal Extraction and isolation
(1) by gained fermentation culture filtered through gauze in embodiment 2, filtrate extracts 3 times by ethyl acetate (volume ratio 1:1), and merge organic layer, cryogenic vacuum concentrate drying obtains brown crude extract F25g;
(2) medicinal extract F is carried out silica gel column chromatography, with mixed organic solvents (chloroform: methyl alcohol, the volume ratio=100:4) wash-out of 10 times of chromatographic column retention volume, merge elutriant, cryogenic vacuum concentrates, and obtains medicinal extract F1;
(3) medicinal extract F1 is carried out silica gel column chromatography, by mixed organic solvents (chloroform: methyl alcohol, volume ratio=100:2) wash-out 5 retention volume, merge elutriant, cryogenic vacuum concentrates, and obtains medicinal extract F2;
(4) dextrane gel Sephadex LH-20 is utilized to carry out column chromatography to medicinal extract F2, eluent is chloroform and the methyl alcohol mixed liquor of volume ratio 1:1, TLC tracing detection contains the part of blue-fluorescence point, collect this elution fractions, after merging elutriant, cryogenic vacuum removing eluent obtains 2,5-diketopiperazines dipeptides (55mg) shown in formula (I).
2, alkaloidal Structural Identification
Target product step 1 obtained carries out mass spectrum and nuclear magnetic resonance spectroscopy.
Mass-spectrometric data is: ESI-MS m/z259 [M-H]
+, 518.8 [2M-H]; Determine molecular formula C
14n
16n
2o
3;
1h and
13c NMR data are in table 1.
Table 1:2,5-diketopiperazines dipeptides (I)
1h spectrum and
13c modal data (500MHz, CDCl
3)
S-is unimodal, d-doublet, m-multiplet
To sum up, the structural formula of described target product is for shown in formula (I):
Isosorbide-5-Nitrae in formula I, 7,9,10,11,12,14 is carbon atom sequence number, this system of compounds called after (3S, 8aS)-3-(4-hydroxybenzyl) hexahydropyrrolo [1,2-a] pyrazine-1,4-dione.
Embodiment 4: dipeptides anti-microbial activity is evaluated
The evaluation of dipeptides anti-microbial activity adopts agar dilution (Letters in Applied Microbiology, 2011,53:546-551), each mensuration in triplicate, test pathogenic bacterium are flavus (Aspergillus flavus) and Candida albicans (Candida albicans), purchased from Beijing North Na Chuanlian Bioteknologisk Institut, strain number is respectively ACCC30321, CAU0037.
Concrete grammar is as follows:
1. bacterium preparation is tested: pathogenic bacterium, after dull and stereotyped activation culture, are forwarded in the triangular flask containing Cha Shi nutrient solution, and under 100 ~ 200rpm, 28 ± 1 DEG C of conditions, shake cultivation 48 ~ 72h, Cha Shi nutrient solution consists of: sucrose 30g/L, NaNO
32.0g/L, K
2hPO
41.0g/L, KCl0.5g/L, MgSO
47H
2o0.5g/L, FeSO
47H
2o(100g/L) 2,1L distilled water, natural ph.2. sample preparation is tested: by dipeptides powder dissolution in dimethyl sulfoxide (DMSO) (DMSO), doubling dilution is adopted to prepare the solution of eight concentration, be respectively 7.5,15,30,60,120,240,480,960 μ g/mL, positive control is amphotericin B, adopt DMSO to dissolve, prepare the solution of the same eight concentration.3. drug sensitive test: each flat board adds 15mL PDA substratum, get 100 μ L Cha Shi nutrient solutions and be spread evenly across in the culture dish containing PDA substratum, PDA substratum consists of: 20g potato, glucose 20g/L, 20g agar, 1L distilled water, natural ph.Getting 20 μ L test samples again joins in culture dish, and even spread, leaves standstill dull and stereotyped 30min, is inverted into by flat board in constant incubator (28 ± 1 DEG C) and cultivates 72h.Take out culture dish, observe and whether have pathogenic bacterium bacterium colony to produce, if not, show pathogenic bacterium suppressed growth under this sample concentration, determine the minimum inhibitory concentration that this sample is antibacterial and MIC value.
Table 2:2,5-diketopiperazines dipeptides (I) and the antibacterial MIC value of amphotericin B (μ g/mL)
Claims (2)
1. 2 shown in preparation formula (I), the method of 5-diketopiperazines dipeptides, described method comprises: will produce the filtering fermentation liquor or centrifugal that the molten bacillus of enzyme (Lysobacter enzymogenes) CCTCC NO:M 2013203 obtains through fermentation, get filtrate or supernatant liquor is extracted with ethyl acetate, get organic layer to concentrate, gained medicinal extract is through separation and purification, obtain described 2, 5-diketopiperazines dipeptides, described separation purification method is as follows: gained medicinal extract is carried out silica gel column chromatography by (1), with chloroform: the solvent of methyl alcohol volume ratio=100:4 is that eluent carries out wash-out, collect elutriant, concentrated, obtain medicinal extract 1, (2) medicinal extract 1 is carried out silica gel column chromatography, with chloroform: the solvent of methyl alcohol volume ratio=100:2 is that eluent carries out wash-out, collect elutriant, concentrated, obtain medicinal extract 2, (3) dextrane gel Sephadex LH-20 is utilized to carry out column chromatography to medicinal extract 2, with chloroform: the solvent of methyl alcohol volume ratio=1:1 is that eluent carries out wash-out, TLC tracing detection contains the part of blue-fluorescence point, collect this elution fractions, after merging elutriant, cryogenic vacuum removes eluent, obtain 2,5-diketopiperazines dipeptides shown in formula (I)
2. produce enzyme molten bacillus (Lysobacter enzymogenes) R-2-1, be preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, preservation date is on May 14th, 2013, and deposit number is CCTCC NO:M 2013203.
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