CN102363797B - Method for producing epsilon-poly-L-lysine - Google Patents
Method for producing epsilon-poly-L-lysine Download PDFInfo
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Abstract
The invention relates to a method for producing epsilon-poly-L-lysine. The method is characterized in that a medium containing 1-10g/L of glycine is added at an early or middle stage of culture. By adding glycine in a fermentation process in the invention, glycine which enters a folic acid metabolism approach allows sufficient one-carbon groups to be supplied to a biosynthesis process, the anabolism activity of bacterial strains to be improved, the synthesis of a precursor L-lysine and epsilon-poly-L-lysine to be increased, and the accumulation amount of epsilon-poly-L-lysine to be 20-50% higher than the accumulation amount obtained through original technologies.
Description
Technical field
The invention belongs to field of fermentation engineering, especially a kind of production method of epsilon-poly-L-lysine.
Background technology
(ε-PL) is the outer linear polymers of a kind of microbial source born of the same parents to epsilon-poly-L-lysine, only is formed by connecting by α-carboxyl and epsilon-amino by 25-35 L-lysine residue.1977, Shima and Sakai found this seed amino acid polymer first in the fermentation clear liquid of Streptomyces albulus NBRC 14147 bacterial strains.Epsilon-poly-L-lysine is a kind of polycation polypeptide nontoxic to human-body safety, can suppress gram negative bacterium, gram-positive microorganism, fungi even some virus, and antimicrobial spectrum is extensive.After by the ε-PL security of mouse experiment proof, Japan, Korea S, the U.S. etc. national with it as antiseptics for natural food and widespread use.ε-PL and derivative range of application thereof are wider, except can be used for food antiseptic, can also be as antiobesity agent, medicine and genophore, retentiveness material, and the Coating Materials in biochip and the biological electronics etc.
According to the retrieval, find following patent documentation about epsilon-poly-L-lysine, wherein CN1260004 discloses a kind of bacterial strain and production method of a large amount of production epsilon-poly-L-lysines, and the method is by aerobic cultivation bacterial strain B21021 (FERM BP-5926) and separation and purification gained epsilon-poly-L-lysine.Bacterial strain B21021 obtains by streptomyces albulus lysinopolymerus subspecies 11011A-1 bacterial strain (FERM BP-1109) is carried out mutagenic treatment, and it is that 10mg/mL or higher S-(2-amino-ethyl)-Cys (AEC) have resistance to concentration.CN101285046 discloses a kind of mutagenic strain TUST2 and has utilized this mutagenic strain to produce the method for epsilon-polylysine and salt thereof, in Chinese Soils of Hainan Province, to separate the TUST1 obtain as starting strain, the bacterial strain TUST2 (CGMCC No.1986) that utilizes the physics and chemistry mutafacient system to select, this bacterial strain has resistance to 10mg/mL or greater concn AEC and 2mg/mL glycine, produces sour amount under the optimal conditions and can reach 10～30g/L.Then fermented liquid obtains the epsilon-polylysine product that molecular weight distribution is 4000～6500Da after the processing such as centrifugal, filtration and weakly acidic cation-exchange resin.CN101525640 discloses a kind of preparation method of epsilon-polylysine, and the method is to utilize streptomyces virginiae Y12, by one-level, secondary seed enlarged culturing, and fermentation production of epsilon-polylysine, yield can reach more than the 8g/L after extracting.Bacterial strain Y12 is that starting strain passes through the mutafacient system acquisitions such as ethyl sulfate, ultraviolet.CN102086441A discloses the brown streptomycete bacterial strain of a kind of ash and has utilized this bacterial strain to prepare the method for epsilon-polylysine and salt thereof, the method is utilized beige streptomycete LS-H1 fermentation production of epsilon-polylysine, accumulation volume is 0.7～20g/L under optimal conditions, and then fermented liquid obtains epsilon-polylysine and salt thereof through centrifugal, ion-exchange.CN101671703 discloses a kind of novel method that improves epsilon-poly-L-lysine output, as producing bacterial strain take Streptomyces diastatochromogenes or Streptomyces albulus, in the process of fermentative production epsilon-poly-L-lysine, product begins after the accumulation, stream adds 1B to improve output in the substratum, obtains the epsilon-poly-L-lysine hydrochloride after then fermented liquid is centrifugal, ion-exchange absorption, decolouring, the vacuum-drying.
Above-mentioned patent document mainly concentrates on the production bacterial classification and zymotechnique of epsilon-poly-L-lysine, but does not all study glycine to the impact of its leavening property, has essential distinction with this patent.
Summary of the invention
The production method that the purpose of this invention is to provide a kind of epsilon-poly-L-lysine, the present invention produces the method for epsilon-poly-L-lysine by increasing the external source medium component, the glycine that contains 1～10g/L in substratum makes the epsilon-poly-L-lysine accumulation volume improve 20～50% than original technique.
The present invention realizes that the technical scheme of purpose is:
A kind of production method of epsilon-poly-L-lysine contains glycine in the substratum.
And the concentration of described glycine is 1～10g/L.
And described glycine is being cultivated the adding of early stage or mid-term.
Advantage of the present invention and positively effect are:
1, the present invention is by adding during the fermentation glycine, glycine can enter the folic acid metabolism approach, for biosynthetic process provides a sufficient carbon-based group, improve bacterial strain anabolism vigor, increase the synthetic of precursor 1B and epsilon-poly-L-lysine, make the epsilon-poly-L-lysine accumulation volume improve 20～50% than original technique.
2, the present invention has changed original zymotechnique, has significantly increased production bacterial strain metabolic activity, has improved the productive rate of epsilon-poly-L-lysine, has increased to a certain extent its accumulation volume, has reduced cost, can be used for plant-scale fermentative production.
Embodiment
The invention will be further described below by specific embodiment, and following examples are descriptive, is not determinate, can not limit protection scope of the present invention with this.
At first need to prove:
1. by the preservation of Chinese common micro-organisms culture presevation administrative center, deposit number is CGMCC No.1986 to the used streptomyces albus (Streptomyces albulus) of following examples, and preservation date is on March 23rd, 2007.
2. the used streptomyces diastatochromogenes (Streptomyces diastatochromogenes) of following examples is by the preservation of Chinese common micro-organisms culture presevation administrative center, deposit number is CGMCC No.3145, and preservation date is on June 29th, 2009.
The present invention illustrates the present invention as an example of above-mentioned two bacterial classifications example, but the scope of used bacterial classification is not limited only to above-mentioned two kinds, all can be applied among the present invention such as the bacterial classification of mentioning in the background technology of the present invention, because principle is identical, concrete operation step is easy to the technician rationally to be known, the present invention gives an example no longer one by one.
During the fermentation stream add 1B be adopt " a kind of novel method that improves epsilon-poly-L-lysine output " (number of patent application is: 200910069517.5, publication number is: the feeding method CN101671703).
4. example 1 and 4 narrations is existing production technique, and embodiment 2 and embodiment 3 are the innovative approach that compares with embodiment 1, and embodiment 5 and embodiment 6 are the innovative approach that compares with embodiment 4.
Embodiment 1:
A kind of production method of epsilon-poly-L-lysine, step is as follows:
In the 100mL substratum, access the spore of 1～2 ring streptomyces diastatochromogenes or streptomyces albus, 30 ℃ of shaking culture 30h; Then in the inoculum size access 100mL fermention medium (initial pH 6.8) with 6% (V/V), cultivate 72h for 30 ℃.In the fermenting process, the epsilon-poly-L-lysine production peak is 0.4g/L.
Embodiment 2:
A kind of production method of epsilon-poly-L-lysine, step is as follows:
In the 100mL substratum, access the spore of 1～2 ring streptomyces diastatochromogenes or streptomyces albus, contain glycine 3g/L in the substratum, 30 ℃ of shaking culture 30h; Then in the inoculum size access 100mL fermention medium (initial pH 6.8) with 6% (V/V), cultivate 72h for 30 ℃.In the fermenting process, the epsilon-poly-L-lysine production peak is 0.5g/L.
Embodiment 3:
A kind of production method of epsilon-poly-L-lysine, step is as follows:
In the 100mL substratum, access the spore of 1～2 ring streptomyces diastatochromogenes or streptomyces albus, 30 ℃ of shaking culture 48h; Then the inoculum size access with 10% (V/V) contains in the 100mL fermention medium of glycine 2g/L, 4.5,30 ℃ of cultivations of initial pH 72h.In the fermenting process, the epsilon-poly-L-lysine production peak is 0.7g/L.
Embodiment 4:
A kind of production method of epsilon-poly-L-lysine, step is as follows:
(1) in the 5L fermentor tank of 2.7L substratum is housed, the seed culture fluid of inoculation 300mL streptomyces diastatochromogenes or streptomyces albus, 30 ℃ of cultivations are treated automatically to be controlled to be 30% when DO is down to 30%, aerobic cultivation 120h, air velocity is 4.0～6.0L/min;
(2) when pH is down to 6.0 left and right sides, (it is 6.0 that the concentration 10～30%g/mL) of ammoniacal liquor is kept pH to Feeding ammonia water, when the remaining sugar concentration in the fermented liquid is down to 10g/L, adds glucose (400g/L) and (NH by stream
4)
2SO
4(80g/L) make remaining sugar concentration reach 12g/L; Simultaneously, no longer control pH, allow it naturally be down to 4.0 and maintain about 4.0;
(3) cultivate 120h, finish fermentation, the highest accumulation epsilon-poly-L-lysine is 11.9g/L in the fermented liquid;
(4) centrifugal removal thalline and part solid substance, utilize the D152 Macroporous weak acid cation exchange resin that supernatant liquor is carried out exchange adsorption and wash-out, elutriant is after the decolouring of D392 macroporous weakly basic anion exchange resin, again after filtration, vacuum concentration, cyclone dryer is dry, obtains the epsilon-poly-L-lysine hydrochloride.
Embodiment 5:
A kind of production method of epsilon-poly-L-lysine, step is as follows:
(1) in the 5L fermentor tank of 2.7L substratum is housed, inoculation contains the seed culture fluid of glycine (3g/L) 300mL streptomyces diastatochromogenes or streptomyces albus, 30 ℃ of cultivations, treat automatically to be controlled to be 30% when DO is down to 30%, aerobic cultivation 120h, air velocity is 4.0～6.0L/min;
(2) when pH is down to 6.0 left and right sides, (it is 6.0 that the concentration 10～30%g/mL) of ammoniacal liquor is kept pH to Feeding ammonia water, when the remaining sugar concentration in the fermented liquid is down to 10g/L, adds glucose (400g/L) and (NH by stream
4)
2SO
4(80g/L) make remaining sugar concentration reach 12g/L; Simultaneously, no longer control pH, allow it naturally be down to 4.0 and maintain about 4.0;
(3) cultivate 120h, finish fermentation, the highest accumulation epsilon-poly-L-lysine 12.9g/L in the fermented liquid;
(4) centrifugal removal thalline and part solid substance, utilize the D152 Macroporous weak acid cation exchange resin that supernatant liquor is carried out exchange adsorption and wash-out, elutriant is after the decolouring of D392 macroporous weakly basic anion exchange resin, again after filtration, vacuum concentration, cyclone dryer is dry, obtains the epsilon-poly-L-lysine hydrochloride.
Embodiment 6:
A kind of production method of epsilon-poly-L-lysine, step is as follows:
(1) in the 5L fermentor tank of 2.7L substratum is housed, inoculation contains the seed culture fluid of glycine (3g/L) 300mL streptomyces diastatochromogenes or streptomyces albus, 30 ℃ of cultivations, treat automatically to be controlled to be 30% when DO is down to 30%, aerobic cultivation 120h, air velocity is 4.0～6.0L/min;
(2) when pH is down to 6.0 left and right sides, (it is 6.0 that the concentration 10～30%g/mL) of ammoniacal liquor is kept pH to Feeding ammonia water, when the remaining sugar concentration in the fermented liquid is down to 10g/L, adds glucose (400g/L) and (NH by stream
4)
2SO
4(80g/L), make remaining sugar concentration reach 12g/L, and stream add 1B (10g/L) in the fermented liquid; Simultaneously, no longer control pH, allow it naturally be down to 4.0 and maintain about 4.0;
(3) cultivate 120h, finish fermentation, the highest accumulation epsilon-poly-L-lysine 15.2g/L in the fermented liquid;
(4) centrifugal removal thalline and part solid substance, utilize the D152 Macroporous weak acid cation exchange resin that supernatant liquor is carried out exchange adsorption and wash-out, elutriant is after the decolouring of D392 macroporous weakly basic anion exchange resin, again after filtration, vacuum concentration, cyclone dryer is dry, obtains the epsilon-poly-L-lysine hydrochloride.
Claims (2)
1. the production method of an epsilon-poly-L-lysine is characterized in that: contain glycine in the substratum; The concentration of described glycine is 2 or 3 g/L, and the bacterial classification of employing is streptomyces albus (Streptomyces albulus) CGMCC No.1986 and streptomyces diastatochromogenes (Streptomyces diastatochromogenes) CGMCC No.3145.
2. the production method of epsilon-poly-L-lysine according to claim 1 is characterized in that: described glycine adds in early stage or mid-term cultivating.
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| CN104004796B (en) * | 2014-04-18 | 2017-06-13 | 天津科技大学 | A kind of fermentation process of the ε polylysines for accumulating homoserine |
| CN104193988B (en) * | 2014-09-01 | 2016-06-29 | 江南大学 | A kind of method of epsilon-polylysine fermentation liquid Flocculation |
| CN105368887B (en) * | 2015-11-05 | 2019-01-22 | 天津科技大学 | A kind of fermentation manufacturing technique of epsilon-poly-L-lysine |
| CN109112169B (en) * | 2017-06-26 | 2021-12-14 | 安泰生物工程股份有限公司 | Polylysine glyceride, preparation method and application thereof, and method for preparing polylysine |
| CN108498401A (en) * | 2018-05-25 | 2018-09-07 | 钱兴 | A kind of preparation method of mouthwash |
| CN111471633B (en) * | 2020-03-13 | 2022-12-06 | 天津科技大学 | Gene engineering high-yield strain streptomyces diastatochromogenes and method for improving yield of epsilon-polylysine |
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| CN101078021A (en) * | 2006-05-22 | 2007-11-28 | 天津科技大学 | Method for producing epsilon-poly-L-lysine by reflux technique |
| CN101285046A (en) * | 2007-04-09 | 2008-10-15 | 天津科技大学 | Mutant strain streptomyces albus TUST2 and process for producing epsilon-polylysine and salts thereof by using the mutant strain |
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| CN101078021A (en) * | 2006-05-22 | 2007-11-28 | 天津科技大学 | Method for producing epsilon-poly-L-lysine by reflux technique |
| CN101285046A (en) * | 2007-04-09 | 2008-10-15 | 天津科技大学 | Mutant strain streptomyces albus TUST2 and process for producing epsilon-polylysine and salts thereof by using the mutant strain |
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Effective date of registration: 20160330 Address after: 317200 No. 18 Feng Dong Road, Fuk Creek Street, Tiantai County, Zhejiang Patentee after: ZHEJIANG SILVER-ELEPHANT BIO-ENGINEERING CO., LTD. Address before: 300457 No. thirteen, 29 Avenue, Tanggu economic and Technological Development Zone, Tianjin Patentee before: Tianjin University of Science & Technology |
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Application publication date: 20120229 Assignee: ZHEJIANG SHENGDA BIO-PHARM Co.,Ltd. Assignor: ZHEJIANG SILVER-ELEPHANT BIO-ENGINEERING Co.,Ltd. Contract record no.: X2021330000154 Denomination of invention: one kind e- Production method of poly-L-lysine Granted publication date: 20130501 License type: Common License Record date: 20210828 |