CN1320099C - Method for preparing epsi-polylysine and its salt by using Kitasatosporia PL6-3 - Google Patents
Method for preparing epsi-polylysine and its salt by using Kitasatosporia PL6-3 Download PDFInfo
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- CN1320099C CN1320099C CNB2005100377742A CN200510037774A CN1320099C CN 1320099 C CN1320099 C CN 1320099C CN B2005100377742 A CNB2005100377742 A CN B2005100377742A CN 200510037774 A CN200510037774 A CN 200510037774A CN 1320099 C CN1320099 C CN 1320099C
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- polylysine
- epsilon
- salt
- kitasatosporia
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Abstract
The present invention discloses a method for preparing epsilon-polylysine and salt thereof by Kitasatospora sp. PL6-3, namely CCTCC No. M205012. The Kitasatospora sp. PL6-3 is a strain obtained by sieving, and is cultivated in a culture medium containing a carbon source, a nitrogen source, etc. so as to accumulate the epsilon-polylysine of 0.4 g/L to 30 g/L under an optimization condition. After thallus in fermentation liquor is removed by centrifugation or filtration, the fermentation liquor is separated to obtain the epsilon-polylysine and the salt thereof by an ion exchange resin method, and the molecular weight is from 3000 Da to 6000 Da. The thallus of the present invention is simple to cultivate, and is favorable for industrial large scale production.
Description
Technical field
The invention belongs to the fermentation engineering field, be specifically related to a kind of high yield epsilon-polylysine (microorganism strains kitasatosporia (Kitasatospora sp.) PL6-3 of ε-PL), and it is used for the production of epsilon-polylysine and salt thereof.
Background technology
(ε-poly-L-lysine, ε-PL) are by microorganism synthetic L-Methionin homopolymer to epsilon-polylysine, are formed by connecting by peptide bond by epsilon-amino and α-carboxyl.Epsilon-polylysine has the activity of resisting gram-positive bacteria, Gram-negative bacteria, fungi and virus, is a kind of biological preservative of excellent property.Except, also as makeup, genophore, medicine encrusting substance, electronic material and environment-friendly materials.
Japanese wine well in 1977 flat one and island clear two find in the streptomyces albus fermented liquid a large amount of D.P. materials is arranged when a large amount of screening D.P. positive materials (alkaloid), this material proved natural ε-PL (Shojishima et al.Agric Biol Chem afterwards, 1977,41 (9): 1807-1809).Subsequently the just great people of grade of Teng Jing found in 1989 Streptomyces noursei also can produce ε-PL (Teng Jing is just great etc. openly specially permit communique [P] .JP401187090A).After this found many bacterial strains that can produce ε-PL, but focused mostly in Streptomycetaceae.Japanese Chisso company utilized streptomyces albus to realize industrialization production in 1989.Because streptomycete is actinomycetes, mycelia is twined comparatively serious, and later stage fermentation liquid is thickness very, and the production difficulty is bigger, and fermentation production rate is very low, and the industrial extensive cheap epsilon-polylysine of producing is still had any problem.For this reason, screen new ε-PL and produce bacterial strain, understand the species diversity that ε-PL produces bacterium, developing efficiently, the epsilon-polylysine preparation method is still very significant.
Summary of the invention
The object of the invention is to provide a kind of new bacterial strain of microorganism of high yield epsilon-polylysine, and it is used for the production of epsilon-polylysine and salt thereof.
The object of the invention can reach by following measures:
The microorganism strains kitasatosporia of seed selection of inventor laboratory and preservation (Kitasatospora sp.) PL6-3, this bacterial strain is preserved in Wuhan China typical culture collection center at present, it abbreviates CCTCC as, the numbering of registering on the books is CCTCC No.M205012, preservation date is: on January 28th, 2005, the depositary institution address is a China. Wuhan. and Wuhan University.With this bacterium as producing bacterial strain.
CCTCC No.M205012 bacterial strain has following character:
1, morphological specificity:
The bacterial strain moderate of on the Gause I substratum, growing, aerial hyphae growth moderate, fibrillae of spores is the curved or Song Rouxuan of ripple, and spore becomes catenation, and the spore oblong is to oval under electron microscope.
2, cultural characteristic:
Cultivated 7-10 days for 30 ℃, observe the feature of bacterial strain on following various substratum.
The cultural characteristic of table 1 PL6-3 bacterial strain
| Substratum | Aerial hyphae | Substrate mycelium | Soluble pigment |
| Gao Shi synthesizes a glucose-asparagine agar glycerine-asparagine agar czapek agar medium inorganic salts starch agar gelatin agar oatmeal agar | Canescence, the relatively poor canescence of growing, well-grown, fine and close canescence, well-grown, compact powdered, white, growth moderate canescence, growth moderate oyster white does not almost have white abundant | The light grey milky white of white milky white～faint yellow white, faint yellow yellowish-brown | Do not have |
3, Physiology and biochemistry character:
(1) culture temperature: at 20-40 ℃.Optimum temperuture is 30 ℃.
(2) gelatine liquefication, starch hydrolysis, milk peptonize: the positive.
(3) milk solidifies: feminine gender.
(4) cellulose utilization: feminine gender.
(5) H
2S generates: feminine gender.
(6) chitinase produces: feminine gender.
(7) pigment produces: produce melanochrome.
(8) anti-NaCl concentration: can grow on 7% concentration.
(9) cell walls is formed: contain LL-DAP, meso-DAP in the full cell hydrolyzed solution of bacterial strain; The sugar composition has rhamnosyl and glucose.
4,16S rDNA sequential analysis:
Record the most of sequence 1227bp of 16S rDNA, (SEQ ID No.1) specific as follows:
5′-ATCAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAA
GCCCTGGAAACGGGGTCTAATACCGGATACGACCTTCCTCCGCATGGGGGTTGGTGGA
AAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGC
CTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTG
AGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGA
AAGCCTGATGCAGCGACGCCGCGTGAGGGACGACGGCCTTCGGGTTGTAAACCTCTTT
CAGCAGGGAAGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCACCGGCTAACTACGT
GCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTATTGGGCGTAAA
GAGCTCGTAGGCGGCCTGTCGCGTCGGATGTGAAAGCCCGGGGCTTAACCCCGGGTCT
GCATTCGATACGGGCAGGCTAGAGTGTGGTAGGGGAGATCGGAATTCCTGGTGTAGCG
GTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCATT
ACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTC
CACGCCGTAAACGTTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAG
CTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAA
TTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAG
AACCTTACCAAGGCTTGACATATGCCGGAAACACCTGGAGACAGGTGCCCCCTTGTGG
TCGGTATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT
CCCGCAACGAGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCTTTCGGGGTGATGGGG
ACTCACAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAATCAT
CATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGTCGGTACAAAGGGCTGCGA
TGCCGCGAGGCGGAGCGAATCCCAAAAAGCCGGCCTCAGTTCGGATTGGGGTCTGCA
ACTCGACCCCATGAAG-3′
The relevant kind of check order row from the GenBank database compared, make up the phylogenetic tree of classifying the basis with 16S rDNA total order as.The result shows: it is all very high that bacterial strain PL6-3 and kitasatosporia belong to (Kitasatospora) homology, with the K.recifensis homology be 98.8%, with the K.kifunensis homology be 98.9%, with the K.sp.C2 homology be 99.3%.The phylogeny result of study of combining form, cytochemistry composition and 16S rDNA complete sequence analysis, the classification position of bacterial strain PL6-3 can be determined to the level of genus, that is: bacterial strain PL6-3 belongs to kitasatosporia and belongs to, and has with kitasatosporia mutation C2 (K.sp.C2) and to be higher than 99% 16S rDNA sequence similarity.
Conclude above-mentionedly, what the present invention used is kitasatosporia, is specially Kitasatospora sp.PL 6-3 (CCTCC No.M205012).
Strain culturing
Utilize kitasatosporia PL6-3 to prepare the method for epsilon-polylysine and salt thereof, with CCTCC No.M205012 bacterial strain slant activation 3 days, 25～37 ℃ of cultivations on the substratum that contains carbon source, nitrogenous source, generate 0.4-30g/L epsilon-polylysine fermented liquid, fermented liquid through centrifugal or remove by filter thalline after, obtain epsilon-polylysine and salt thereof by the ion-exchange-resin process separation, its molecular weight is at 3000-6000Da.
Nutritional factor such as the substratum carbonaceous sources that the present invention uses, nitrogenous source, inorganic salt.
The present invention does not limit carbon source is special, can use glucose, sucrose, amylum hydrolysate of the sugar, glycerine, maltose, wood sugar, pectinose, semi-lactosi, raffinose, molasses etc., and is wherein more suitable with glucose.
Nitrogenous source comprises organic nitrogen source such as peptone, extractum carnis, yeast extract paste, urea, corn steep liquor, soya-bean cake hydrolyzed solution, cotton seed powder cake, also can adopt inorganic nitrogen-sourced as (NH
4)
2SO
4, NH
4NO
3, NH
4Cl, above-mentioned nitrogenous source may be used alone, can also be used in combination.
It is to adopt ion exchange method that epsilon-polylysine that the present invention adopts and salt thereof separate purifying.Utilize method centrifugal or the flocculation thalline to remove thalline in the fermented liquid, supernatant liquor carries out ion exchange resin treatment (ion exchange resin treatment is that Zeo-karb is handled) then, carry out concentrating under reduced pressure, activated carbon decolorizing to collecting liquid, next utilize lower alcohols such as ethanol and rudimentary ethers such as ether, precipitation obtains epsilon-polylysine and salt thereof.
The epsilon-polylysine and the salt thereof of the present invention's preparation have following physico-chemical property:
(1) water-soluble, the hydrochloric acid of this product is slightly soluble in ethanol, is insoluble to organic solvents such as ether, ethyl acetate;
(2) ninhydrin reaction is positive, with after the 6N HCl hydrolysis triketohydrindene hydrate being positive.
(3) after the 6N HCl hydrolysis, detect with thin-layer chromatography, find to generate single amino acid-Methionin in the hydrolyzed solution, this product is the high molecular polymer of Methionin.
(4) adopt the Sanger method, the structure of having identified this product is ε-type structure, by the ε-NH of a L-Methionin
2The polymer epsilon-polylysine that is connected with peptide bond that the α of another L-Methionin-COOH forms and aggregates into.
(5) measuring ε-PL molecular weight by the SDS-PAGE electrophoresis is 3000-5000Da.
The epsilon-polylysine and the salt thereof of the present invention's preparation has high-cation, the biocidal property of wide spectrum is arranged, owing to be L-Methionin homopolymer, after human body is edible, degradable is this essential amino acid of L-Methionin, be further used for protein synthesis or continue metabolism, do not have any toxicity, so it is described as " nutritional type sanitas ".The epsilon-polylysine and the salt thereof of the present invention's preparation also have high-hydroscopicity etc., are expected to be widely used in aspects such as medical material, makeup, hairdressing agent, fodder additives, agricultural chemicals, foodstuff additive, electronic material, high absorbency material and engineering carrier material.
Advantage of the present invention:
The microorganism strains that the present invention screens can be produced epsilon-polylysine and salt thereof, and the fermenting process mycelia fragments into rod-short, does not have wrapping phenomena to take place, and operates more conveniently, and culture condition is very extensive.
Description of drawings
Fig. 1 is the mycelia photo of strain growth of the present invention on solid medium.
Fig. 2 is the fibrillae of spores photo of strain growth of the present invention on solid medium.
Embodiment
Below be embodiment, the invention will be further described for the general, but to the present invention without limits.
Embodiment 1
Slant medium: Glucose 1%, extractum carnis 0.5%, yeast extract paste 0.5%, K
2HPO
40.1%, MgSO
4.7H
2O 0.05%, agar 2%, pH7.0
Shake-flask culture base: Glucose 5%, (NH
4)
2SO
41%, Na
2HPO
4.12H
2O 0.158%, KH
2PO
4.7H
2O 0.136%, MgSO
4.7H2O 0.05%, ZnSO
4.7H2O 0.004%, FeSO
4.7H
2O0.003%, yeast extract paste 0.5%, pH6.8.Dress liquid 50ml sterilized 15 minutes for 121 ℃ in the 250ml volumetrical triangular flask.
With kitasatosporia PL6-3 (CCTCC No.M205012) 28-32 ℃ of cultivation 72h on slant medium of purifying, the spore that connects this bacterium of ring is then cultivated 72h, is shaken a bottle rotating speed 200r/min for 30 ℃ in the shake-flask culture base.Obtaining fermented liquid epsilon-polylysine content at last is 0.944g/L.
Embodiment 2
With glucose 150g, yeast extract paste 15g, (NH
4)
2SO
430g, MgSO
4.7H
2O 0.75g, FeSO
4.7H
2O0.09g, ZnSO
4.7H
2O0.12g, KH
2PO
4.7H
2O4.08g, Na
2HPO
4.12H
2O4.74g transfers pH6.8 with NaOH, and the water constant volume is to 3L, the 5 liters of glass stirred fermentors of packing into, 121 ℃ of steam sterilizings 15 minutes.K.sp.PL6-3 (CCTCC No.M205012) is cultivated 24h for 30 ℃ with seed culture medium, and seed culture medium is identical with fermention medium.Seed liquor 300ml is inserted in the cooled fermented liquid 30 ℃ of cultivations, (ventilating ratio 1: 2.5vvm, stirring velocity is 350r/min), along with incubation time prolongs, the epsilon-polylysine in the nutrient solution is on the increase, during to 72h, contain epsilon-polylysine 3.0g/L in the fermented liquid.
Embodiment 3
With embodiment 2, fermenting process control pH4.0 adds glucose and nitrogenous source, to keep about glucose concn 10g/L fermentation 168h, accumulation epsilon-polylysine 25g/L in the fermented liquid.With the centrifugal removal thalline of fermented liquid, 732 cationic exchange resin adsorption on the supernatant liquor, washing, 1N ammoniacal liquor wash-out is collected liquid and is concentrated except that ammonia, and activated carbon decolorizing continues to concentrate the back and uses ethanol sedimentation, obtains epsilon-polylysine hydrochloride 15.25g.The specific rotatory power of product
(c, 1H
2O), to record molecular weight be 5000Da to the SDS-PAGE electrophoresis.
Sequence table
＜110〉Nanjing University of Technology
＜120〉utilize kitasatosporia PL6-3 to prepare the method for epsilon-polylysine and salt thereof
<160>1
<210>1
<211>1227
<212>DNA
＜213〉kitasatosporia (Kitasatospora sp.)
<400>1
atcagtggcg aacgggtgag taacacgtgg gcaatctgcc ctgcactctg ggacaagccc 60
tggaaacggg gtctaatacc ggatacgacc ttcctccgca tgggggttgg tggaaagctc 120
cggcggtgca ggatgagccc gcggcctatc agcttgttgg tggggtaatg gcctaccaag 180
gcgacgacgg gtagccggcc tgagagggcg accggccaca ctgggactga gacacggccc 240
agactcctac gggaggcagc agtggggaat attgcacaat gggcgaaagc ctgatgcagc 300
gacgccgcgt gagggacgac ggccttcggg ttgtaaacct ctttcagcag ggaagaagcg 360
caagtgacgg tacctgcaga agaagcaccg gctaactacg tgccagcagc cgcggtaata 420
cgtagggtgc gagcgttgtc cggaattatt gggcgtaaag agctcgtagg cggcctgtcg 480
cgtcggatgt gaaagcccgg ggcttaaccc cgggtctgca ttcgatacgg gcaggctaga 540
gtgtggtagg ggagatcgga attcctggtg tagcggtgaa atgcgcagat atcaggagga 600
acaccggtgg cgaaggcgga tctctgggcc attactgacg ctgaggagcg aaagcgtggg 660
gagcgaacag gattagatac cctggtagtc cacgccgtaa acgttgggaa ctaggtgttg 720
gcgacattcc acgtcgtcgg tgccgcagct aacgcattaa gttccccgcc tggggagtac 780
ggccgcaagg ctaaaactca aaggaattga cgggggcccg cacaagcagc ggagcatgtg 840
gcttaattcg acgcaacgcg aagaacctta ccaaggcttg acatatgccg gaaacacctg 900
gagacaggtg cccccttgtg gtcggtatac aggtggtgca tggttgtcgt cagctcgtgt 960
cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgttctgtgt tgccagcatg 1020
cctttcgggg tgatggggac tcacaggaga ctgccggggt caactcggag gaaggtgggg 1080
acgacgtcaa atcatcatgc cccttatgtc ttgggctgca cacgtgctac aatggtcggt 1140
acaaagggct gcgatgccgc gaggcggagc gaatcccaaa aagccggcct cagttcggat 1200
tggggtctgc aactcgaccc catgaag 1227
Claims (5)
1, a kind of kitasatosporia (Kitasatospora sp) PL6-3 is by China's typical culture collection center preservation, preserving number CCTCC No.M205012.
2, utilize kitasatosporia PL6-3 to prepare the method for epsilon-polylysine and salt thereof, it is characterized in that 25～37 ℃ of cultivations on the substratum that contains carbon source, nitrogenous source of CCTCC No.M205012 bacterial strain, generate the epsilon-polylysine fermented liquid, fermented liquid through centrifugal or remove by filter thalline after, obtain epsilon-polylysine by the ion-exchange-resin process separation, its molecular weight is at 3000-6000Da.
3, method according to claim 2, the carbon source that it is characterized in that substratum is glucose, sucrose, amylum hydrolysate of the sugar, glycerine, maltose, wood sugar, pectinose, semi-lactosi, raffinose or molasses.
4, method according to claim 2 is characterized in that the nitrogenous source of substratum adopts organic nitrogen source extractum carnis, yeast extract paste, peptone, corn steep liquor, urea, soya-bean cake hydrolyzed solution, cotton seed powder cake; Perhaps adopt inorganic nitrogen-sourced (NH
4)
2SO
4, NH
4Cl, NH
4NO
3Above-mentioned nitrogenous source uses separately or compound use.
5, method according to claim 2 is characterized in that the ion exchange resin that adopts is Zeo-karb.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101580865B (en) * | 2008-05-15 | 2012-09-26 | 上海医药工业研究院 | Method for sieving Epsilon-polylysine high-producing strains |
| CN104193988B (en) * | 2014-09-01 | 2016-06-29 | 江南大学 | A kind of method of epsilon-polylysine fermentation liquid Flocculation |
| CN105112301A (en) * | 2015-09-06 | 2015-12-02 | 樟树市狮王生物科技有限公司 | Natural preservative epsilon-polylysine strain and methods for screening, fermenting and identifying same |
| CN105053517B (en) * | 2015-09-21 | 2018-09-28 | 山东西王糖业有限公司 | A kind of production method of nonreactive feed |
| CN107164417A (en) * | 2017-05-17 | 2017-09-15 | 郑州拜纳佛生物工程股份有限公司 | A kind of production method of ε polylysines |
| CN116855416B (en) * | 2023-07-13 | 2023-12-12 | 贵州省茶叶研究所 | Bacterial strain for antagonizing plant disease pathogenic bacteria, microbial inoculum and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1036955A (en) * | 1988-03-11 | 1989-11-08 | 科研制药株式会社 | Substance for agricultural use and preparation method thereof |
| CN1529712A (en) * | 2001-07-26 | 2004-09-15 | ֮����ʽ���� | Process for preparing dipeptide, Peptide synthase to be used therein and process for producing peptide synthase |
-
2005
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1036955A (en) * | 1988-03-11 | 1989-11-08 | 科研制药株式会社 | Substance for agricultural use and preparation method thereof |
| CN1529712A (en) * | 2001-07-26 | 2004-09-15 | ֮����ʽ���� | Process for preparing dipeptide, Peptide synthase to be used therein and process for producing peptide synthase |
Non-Patent Citations (1)
| Title |
|---|
| 微生物产生的酶抑制剂 刘华珍等，抗生素，第8卷第1期 1983 * |
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Application publication date: 20051116 Assignee: ZHANGSHU LIONKING BIOLOGICAL TECHNOLOGY CO., LTD. Assignor: Nanjing University of Technology Contract record no.: 2013320000330 Denomination of invention: Method for preparing epsilon lysine and its salt by using PL6-3 of northern spore fungus Granted publication date: 20070606 License type: Exclusive License Record date: 20130415 |
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