CN104004796A - Epsilon-polylysine fermentation method by homoserine accumulation - Google Patents
Epsilon-polylysine fermentation method by homoserine accumulation Download PDFInfo
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Abstract
The invention relates to an eapsilon-polylysine (eapsilon-PL) fermentation method by homoserine accumulation. Streptomyces diastatochromogenes CGMCCNo.3145 is adopted as a production strain, and after 0-48h fermentation, L-threonine with a final concentration of 2.5-5.0g/L is added into a fermentation medium. The involved adding of L-threonine, L-methionine and L-leucine to the fermentation liquid in the invention is different from of other amino acid adding for improving the eapsilon-PL yield, and improvement of the eapsilon-PL yield is realized by inhibiting shunt metabolism and changing metabolic flux distribution. Under the same raw material input, a higher product concentration can be acquired, the by-product concentration is decreased, and purification is easy.
Description
Technical field
The invention belongs to biological technical field, relate to the production method of epsilon-polylysine (ε-PL), especially a kind of fermentation process of the epsilon-polylysine that accumulates homoserine.
Background technology
ε-PL is the homopolymer that contains 25-35 L-Lys residue being produced by actinomycetes, and these L-Lys residues are connected with epsilon-amino by α-carboxyl.ε-PL antimicrobial spectrum is comparatively extensive, and gram-positive microorganism, Gram-negative bacteria, yeast and mould etc. are all had to restraining effect in various degree.For most of bacterium, its minimal inhibitory concentration (MIC) is 1-8g/L(checking data); And for yeast and fungi, its MIC is more higher a little.500mg/L can make long non-contractile tail phage inactivation.ε-PL can be decomposed into one of eight kinds of indispensable amino acids of L-Lys(in human body), and the latter Ye Shi various countries allow one of amino acid of strengthening in food.Therefore, ε-PL is a kind of nutritional type fungistat, safe in Chemical Preservative, meanwhile, considers its security and biodegradability, and within 1989, Japan ratifies it for processed food, and Korea S and the U.S. also allow it to use as foodstuff additive subsequently.In January, 2004, U.S. food and drug administration (FDA) assert that ε-PL is " it is generally acknowledged safe " (Generally RecognizedasSafe, GRAS).2010, FDA issued the bulletin authenticating by GRAS about ε-PL, in bulletin, pointed out, ε-PL can be used as food preservatives and is applied to all kinds of traditional foods under given conditions.
As far back as 1977, the Shima and Sakai of Japan found that S.albulus NBRC 14147 can synthesize ε-PL.Afterwards, have much about the report that improves ε-PL output.The people such as Hirohara report in the article of delivering for 2006, in USE-51 fermenting process, supplement glycerine with constant speed in substratum, can improve acid yield, and maintain the stability of pH.They study the process of bacterial strain USE-11 and USE-51 product ε-PL, add the organic acid in TCA circulation, citric acid, succsinic acid, α-ketoglutaric acid and oxysuccinic acid, find that citric acid can significantly improve the output of ε-PL, succsinic acid suppresses the synthetic of ε-PL completely, and α-ketoglutaric acid and oxysuccinic acid produce sour impact to USE-11 and USE-51 and fall between.This phenomenon may be to generate L-Aspartic acid because Citrate trianion can promote oxaloacetic acid to transform, and the accumulation of L-Aspartic acid causes a large amount of synthetic of 1B.α-ketoglutaric acid shows the impact in fermenting process, and TCA circulation may divide and forms Pidolidone salt, so that these substances are combined with L-Aspartic acid-beta galactose, and generation diaminopimelic acid intermediate (Fig. 1).Oxysuccinic acid shows the impact of fermentation, and this organic acid can improve the synthetic of L-Aspartic acid and Pidolidone.Shima etc. think that L-Lys molecule can directly be utilized by ε-PL biosynthesizing.Therefore, the people such as Hirohara are in the fermenting process of USE-51, and medium component includes citric acid, glycerine and (NH
4)
2sO
4time, to the L-lysine that adds 11mM in substratum, can increase substantially the output of ε-PL.
Through retrieval, find the patent documentation of producing about ε-PL as follows, concrete disclosure is:
1, producing in a large number the bacterial strain of epsilon-poly-L-lysine and production method (CN1260004) discloses a kind of more conventional raising bacterial strain and has produced the throughput of ε-PL and the method for scale operation ε-PL.
2, a kind of mutagenic strain streptomyces albus TUST2 and the method (CN101285046) of utilizing this mutagenic strain to produce epsilon-polylysine and salt thereof disclose and have a kind ofly combined with ultraviolet mutagenesis, ultraviolet and chemomorphosis, and the means mutagenesis streptomyces albus TUST2 such as N ion implantation mutagenesis and utilize the method for this mutagenic strain production epsilon-polylysine and salt thereof.
3, utilizing glycerine is that method (CN102352385A) that single carbon source through fermentation is produced epsilon-polylysine discloses employing glycerine and makes a carbon source, adds fermentation process production epsilon-polylysine in conjunction with two stage pH value regulating strategies and stream.
4, utilize the method (CN102352386A) of glucose and glycerine mixed carbon source fermentation production of epsilon-polylysine to disclose employing glucose and glycerine jointly as a carbon source, add fermentation process and pH control strategy is produced epsilon-polylysine in conjunction with stream.
5, a kind of novel method (CN101671703) that improves epsilon-poly-L-lysine output discloses and has utilized glucose to make carbon source and stream adds 1B fermentation production of epsilon-PL.
6, a kind of production method of epsilon-poly-L-lysine (CN102363797A) discloses by adding during the fermentation glycine, glycine can enter 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 epsilon-poly-L-lysine accumulation volume improve 20～50% than original technique.
7, promote the synthetic method of epsilon-polylysine (CN103333926A) disclose by epsilon-polylysine fermenting process in batches or continuously stream add described Pidolidone and/or Pidolidone sodium solid or a solution, realize epsilon-polylysine output and significantly improve.
It is 8, a kind of that to utilize method (CN1696279) that kitasatosporia PL6-3 prepares epsilon-polylysine and salt thereof to disclose a kind of kitasatosporia PL6-3 obtaining by screening be the method that CCTCCNo.M205012 prepares epsilon-polylysine and salt thereof.
In report and patent documentation before, about improving ε-PL aspect, by adding the organic acid in TCA circulation or adding 1B, L-glycine, L-Glu and/or Pidolidone sodium and realize, these organic acids or amino acid are all direct or indirect precursor substances of the synthetic ε-PL of streptomycete, and L-threonine, L-Methionine and L-Leu are different from above-mentioned a few seed amino acid.As can be seen from Figure 2, epsilon-polylysine route of synthesis also relates to other amino acid whose synthesizing (as L-Methionine, L-Leu and L-threonine), E.C. 2.7.2.4. (Ask in approach, catalysis L-Aspartic acid generates L-aspartylphosphate) and homoserine dehydrogenase (Hsd is reduced to homoserine by aspartic-β-semialdehyde in the presence of NAD or NADP) can be subject to the amino acid whose regulation and control of end product.With respect to 1B, the synthetic shunt metabolism that belongs to of L-threonine, L-Methionine and L-Leu.
Summary of the invention
The object of this invention is to provide a kind of fermentation process of the epsilon-polylysine that accumulates homoserine, the present invention adds L-threonine in epsilon-polylysine fermenting process, regulates and controls epsilon-polylysine producing strains metabolism, improves the method for output.
The technical scheme that the present invention realizes is:
A kind of fermentation process of the epsilon-polylysine that accumulates homoserine, adopt streptomyces diastatochromogenes (Streptomyces diastatochromogenes) CGMCCNo.3145 as producing bacterial strain, strain fermentation 0-48h, in fermented liquid, add L-threonine, improve the output of epsilon-polylysine;
Described fermention medium is M3G substratum g/L: glucose 50, yeast powder 5, (NH
4)
2sO
410, KH
2pO
41.36, K
2hPO
40.8, MgSO
47H
2o0.5, ZnSO
47H
2o0.04, FeSO
47H
2o0.03;
By the by volume inoculum size of mark 6-10% of seed liquor, switching seed liquor is fermented to being equipped with in M3G medium container, at fermentation 0-48h, in substratum, adds the L-threonine that final concentration is 2.5-5.0g/L.
And the preparation method of the seed liquor of described streptomyces diastatochromogenes is: by after slant strains activation, get 1-3 ring, be seeded in the triangular flask that M3G substratum is housed, 28-37 DEG C, 150-200rpm/min, cultivates 24-48h, obtains seed liquor.
And described fermentation process is shake flask fermentation or the fermentation of ventilation stirred-tank fermenter.
And described ventilation stirred-tank fermenter ferments during the fermentation, pH controls and is divided into two stages, and the glucose content in substratum is higher than 10g/L, and pH value maintains 6.0-6.5; Glucose content in substratum lower than 10g/L after, pH value maintains 3.5-4.0.
And, when the glucose concn in described fermention medium is during lower than 10g/L, adding glucose 400g/L, ammonium sulfate 80g/L by self-feeding pump stream, the final concentration of glucose maintains 8-13g/L.
Advantage of the present invention and positively effect are:
1, in fermented liquid, add L-threonine, L-Methionine and the L-Leu that in the present invention, relate to are different from other amino acid whose interpolations and improve ε-PL output, but change metabolic flux analysis and realize and improve ε-PL output by suppressing shunt metabolism, that same materials drops into the higher production concentration of acquisition, by-product concentration reduces, and purifying is simple.
2, the present invention causes the accumulation of L-homoserine according to the excessive meeting of the L-threonine in fermented liquid, L-Methionine and L-Leu, thereby metabolism stream flows to and changes, more flow direction synthesized 1B approach, thereby improves ε-PL output, and maximum improves 188%.
3, the present invention's strict relation of controlling pH value and glucose in production stage, makes the fermentation capacity of bacterial classification perform to maximum value, has obtained maximum epsilon-polylysine output.
Brief description of the drawings
Fig. 1 is the possible route of synthesis of ε-PL in Streptomyces of the present invention, from glucose;
Fig. 2 is the possible route of synthesis of ε-PL in Streptomyces of the present invention, from aspartic acid.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
A kind of fermentation process of the epsilon-polylysine that accumulates homoserine, shown in the bacterial classification used be streptomyces diastatochromogenes (Streptomyces diastatochromogenes) CGMCCNo.3145, this bacterial strain is by China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, deposit number is CGMCCNo.3145, and preservation date is on June 29th, 2009.
The substratum using is:
Slant medium: take 20g soybean cake powder, be dissolved in 1L tap water, 121 DEG C of heating 30min.Then, filter with 8 layers of gauze; In gained filtrate, add 20g N.F,USP MANNITOL, be adjusted to pH7.0 with 2MNaOH; Finally, add 2% agar powder, and be settled to 1L, 121 DEG C of sterilizing 20min with tap water.
Fermention medium (g/L): glucose 50, yeast powder 5, (NH4) 2SO45, KH2PO41.36, K2HPO40.8, FeSO
47H
2o0.03, is adjusted to pH7.2,121 DEG C of sterilizing 20min with ammoniacal liquor.
Accumulate a fermentation process for the epsilon-polylysine of homoserine, specific as follows:
(1) slant strains preparation: by streptomyces diastatochromogenes CGMCCNo.3145 streak inoculation on the slant medium preparing, be placed in 30 DEG C of constant incubators, cultivate 5-7 days, media surface generates grey spore, puts into 4 DEG C of preservations of refrigerator, for subsequent use.
(2) the preparation of seed liquor: the slant strains of 4 DEG C of preservations of refrigerator is activated to 3-4h at 30 DEG C, transfering loop is got 1-3 ring, is inoculated into (liquid amount is the 10-20% of triangular flask volume) in the triangular flask that fermention medium is housed 28-37 DEG C, 150-200rpm/min, cultivates 24-48h.
(3) shake flask fermentation: by cultured seed liquor according to 6%-10%(v/v) inoculum size, be seeded to (liquid amount is the 10-20% of triangular flask volume) in the triangular flask that fermention medium is housed, add the L-threonine of 2.5-5g/L at fermentation 0-48h, 28-37 DEG C, 150-200rpm/min, cultivates 72-120h.
(4) the stirred-tank fermenter that ventilates fermentation: add fermention medium in ventilation stirred-tank fermenter, inoculum size 6%-10%(V/V), mixing speed 300-750rpm/min, air flow 3.0 ± 0.5vvm, make Dissolved Oxygen concentration Control 30%, temperature 30-37 DEG C, cultivates 120-188h, pH adopts pH electrode to detect online, and adding 50% ammoniacal liquor by self-feeding pump stream, to control pH(specifically how many).The interpolation of L-threonine, by being configured to certain density mother liquor, at fermentation 0-48h, utilizes self-feeding pump stream to be added in fermentor tank, and the final concentration of L-threonine is 2.5-5g/L.
During the fermentation, pH controls and is divided into two stages, and at first stage (glucose content in substratum is higher than 10g/L), pH value maintains 6.5, to accumulate a large amount of thalline; At subordinate phase (glucose content in substratum lower than 10g/L after), pH value maintains 3.0-3.5, to produce a large amount of ε-PL, every 2h gets sample one time, in subordinate phase, when the glucose concn in substratum is during lower than 10g/L, add glucose (400g/L), ammonium sulfate (80g/L) by self-feeding pump stream, the final concentration of glucose maintains 8-13g/L.
(5) in fermenting process, the detection of L-homoserine in born of the same parents: after fermentation ends, get a certain amount of thalline, centrifuge washing, uses liquid nitrogen smudge cells, and cold methanol extracts born of the same parents' intracellular metabolite thing, detects for GC-MS.Wherein, oxaloacetic acid (0.14g/L) is the quantitative reference substance as L-homoserine as internal standard substance, and the L-homoserine detecting is relative concentration.
In order to describe aforesaid method in detail, provide following examples.
Embodiment 1
Streptomyces diastatochromogenes CGMCCNo.3145 shake flask fermentation
By seed liquor according to inoculum size 6%(V/V) be forwarded in the 500mL triangular flask that 100mL fermention medium is housed and carry out shake flask fermentation, at the initial 0h of fermentation, in substratum, add the L-threonine that final concentration is 2.5g/L, 30 DEG C, 180rpm/min, fermentation 72h, in fermented liquid, ε-PL output is 0.750g/L, the output (0.463g/L) of not adding L-threonine control group has improved 61%, in born of the same parents, the content of L-homoserine is 2.93mg/L, does not add L-threonine control group and improves (1.51mg/L) raising 94%.
Embodiment 2
Streptomyces diastatochromogenes CGMCCNo.3145 shake flask fermentation
By seed liquor according to inoculum size 10%(V/V) be forwarded in the 1000mL triangular flask that 200mL fermention medium is housed and carry out shake flask fermentation, fermentation 0h, in substratum, add the L-threonine that final concentration is 5g/L, fermentation 72h, in fermented liquid, ε-PL output is 0.992g/L, the output (0.463g/L) of not adding L-threonine control group has improved 114%, and in born of the same parents, the content of L-homoserine is 2.83mg/L, does not add L-threonine control group and improves (1.58mg/L) raising 79%.
Embodiment 3
Streptomyces diastatochromogenes CGMCCNo.3145 shake flask fermentation
By seed liquor according to inoculum size 6%(V/V) be forwarded in the 500mL triangular flask that 100mL fermention medium is housed and carry out shake flask fermentation, fermentation 24h, in substratum, add the L-threonine that final concentration is 5g/L, fermentation 72h, in fermented liquid, ε-PL output is 0.886g/L, the output (0.463g/L) of not adding L-threonine control group has improved 91%, and in born of the same parents, the content of L-homoserine is 2.70mg/L, does not add L-threonine control group and improves (1.66mg/L) raising 63%.
Embodiment 4
The fermentation of streptomyces diastatochromogenes CGMCCNo.3145 ventilation stirred-tank fermenter
By seed liquor according to inoculum size 10%(V/V) be forwarded in the 5L ventilation stirred-tank fermenter that 3L fermention medium is housed, fermentation 0h, in substratum, add the L-threonine that final concentration is 5g/L, mixing speed 300-750rpm/min, air flow 3.0 ± 0.5vvm, makes Dissolved Oxygen concentration Control 30%, 30 DEG C of temperature, pH adopts pH electrode to detect online, adds 50% ammoniacal liquor control pH by self-feeding pump stream.During the fermentation, pH controls and is divided into two stages, and at first stage (glucose content in substratum is higher than 10g/L), pH value maintains 6.0-6.5, to accumulate a large amount of thalline; At subordinate phase (glucose content in substratum lower than 10g/L after), pH value maintains 3.0-3.5, to produce a large amount of ε-PL.Every 2h gets sample one time.In subordinate phase, add glucose (400g/L), ammonium sulfate (80g/L) by self-feeding pump stream, the final concentration of glucose maintains 8-13g/L, ferments to 188h.
After testing, ε-PL output in fermented liquid is 34.63g/L, under same culture conditions, the output (11.99g/L) of not adding L-threonine control group has improved 188%, in born of the same parents, the content of L-homoserine is 2.60mg/L, does not add L-threonine control group and improves (1.37mg/L) raising 89%.
Embodiment 5
The fermentation of streptomyces diastatochromogenes CGMCCNo.3145 ventilation stirred-tank fermenter
By seed liquor according to inoculum size 10%(V/V) be forwarded in the 20L ventilation stirred-tank fermenter that 13L fermention medium is housed, fermentation 48h, in substratum, add the L-threonine that final concentration is 5g/L, mixing speed 300-750rpm/min, air flow 3.0 ± 0.5vvm, makes Dissolved Oxygen concentration Control 30%, 30 DEG C of temperature, pH adopts pH electrode to detect online, adds 50% ammoniacal liquor control pH by self-feeding pump stream.
During the fermentation, pH controls and is divided into two stages, and at first stage (glucose content in substratum is higher than 10g/L), pH value maintains 6.3-6.6, to accumulate a large amount of thalline; At subordinate phase (glucose content in substratum lower than 10g/L after), pH value maintains 3.0-3.5, to produce a large amount of ε-PL.
Every 2h gets sample one time.In subordinate phase, add glucose (400g/L), ammonium sulfate (80g/L) by self-feeding pump stream, the final concentration of glucose maintains 8-13g/L, ferments to 164h.
After testing, ε-PL output in fermented liquid is 25.47g/L, under identical fermentation condition, the output (11.99g/L) of not adding L-threonine control group has improved 112%, in born of the same parents, the content of L-homoserine is 2.13mg/L, does not add L-threonine control group and improves (1.12mg/L) raising 90%.
Claims (5)
1. one kind accumulates the fermentation process of the epsilon-polylysine of homoserine, it is characterized in that: adopt streptomyces diastatochromogenes (Streptomyces diastatochromogenes) CGMCCNo.3145 as producing bacterial strain, strain fermentation 0-48h, in fermention medium, add L-threonine, improve the output of epsilon-polylysine;
Described fermention medium (g/L): glucose 50, yeast powder 5, (NH
4)
2sO
45, KH
2pO
41.36, K
2hPO
40.8, FeSO
47H
2o0.03;
By the by volume inoculum size of mark 6-10% of seed liquor, switching seed liquor is fermented to being equipped with in fermentation culture based containers, at fermentation 0-48h, in substratum, adds the L-threonine that final concentration is 2.5-5.0g/L.
2. the fermentation process of the epsilon-polylysine of accumulation homoserine according to claim 1, it is characterized in that: the preparation method of the seed liquor of described streptomyces diastatochromogenes is: by after slant strains activation, get 1-3 ring, be seeded in the triangular flask that fermention medium is housed, 28-37 DEG C, 150-200rpm/min, cultivates 24-48h, obtains seed liquor.
3. the fermentation process of the epsilon-polylysine of accumulation homoserine according to claim 1, is characterized in that: described fermentation process is shake flask fermentation or the fermentation of ventilation stirred-tank fermenter.
4. the fermentation process of the epsilon-polylysine of accumulation homoserine according to claim 3, it is characterized in that: described ventilation stirred-tank fermenter ferments during the fermentation, pH controls and is divided into two stages, and the glucose content in substratum is higher than 10g/L, and pH value maintains 6.5; Glucose content in substratum lower than 10g/L after, pH value maintains 3.0-3.5.
5. the fermentation process of the epsilon-polylysine of accumulation homoserine according to claim 4, it is characterized in that: when the glucose concn in described fermention medium is during lower than 10g/L, add glucose 400g/L, ammonium sulfate 80g/L by self-feeding pump stream, the final concentration of glucose maintains 8-13g/L.
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Cited By (6)
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| CN104726509A (en) * | 2015-02-09 | 2015-06-24 | 苏州科技学院 | Method for producing epsilon-polylysine through fermentation of cassava starch |
| CN105368887A (en) * | 2015-11-05 | 2016-03-02 | 天津科技大学 | Fermentation production process of Epsilon-poly-L-lysine |
| CN111471633A (en) * | 2020-03-13 | 2020-07-31 | 天津科技大学 | Gene engineering high-yield strain streptomyces diastatochromogenes and method for improving yield of polylysine |
| CN111607608A (en) * | 2020-04-20 | 2020-09-01 | 天津科技大学 | Gene engineering high-yield strain streptomyces diastatochromogenes, method for improving yield of polylysine and application |
| CN111621454A (en) * | 2020-04-20 | 2020-09-04 | 天津科技大学 | Production method and application of genetic engineering high-yield strain streptomyces diastatochromogenes and polylysine |
| CN113897301A (en) * | 2021-05-31 | 2022-01-07 | 天津科技大学 | Gene engineering high-yield strain streptomyces diastatochromogenes, production method and application of epsilon-polylysine |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726509A (en) * | 2015-02-09 | 2015-06-24 | 苏州科技学院 | Method for producing epsilon-polylysine through fermentation of cassava starch |
| CN105368887A (en) * | 2015-11-05 | 2016-03-02 | 天津科技大学 | Fermentation production process of Epsilon-poly-L-lysine |
| 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 |
| CN111471633A (en) * | 2020-03-13 | 2020-07-31 | 天津科技大学 | Gene engineering high-yield strain streptomyces diastatochromogenes and method for improving yield of polylysine |
| 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 |
| CN111607608A (en) * | 2020-04-20 | 2020-09-01 | 天津科技大学 | Gene engineering high-yield strain streptomyces diastatochromogenes, method for improving yield of polylysine and application |
| CN111621454A (en) * | 2020-04-20 | 2020-09-04 | 天津科技大学 | Production method and application of genetic engineering high-yield strain streptomyces diastatochromogenes and polylysine |
| CN113897301A (en) * | 2021-05-31 | 2022-01-07 | 天津科技大学 | Gene engineering high-yield strain streptomyces diastatochromogenes, production method and application of epsilon-polylysine |
| CN113897301B (en) * | 2021-05-31 | 2023-10-27 | 天津科技大学 | Production method and application of genetically engineered high-yield strain streptomyces diastatochromogenes and epsilon-polylysine |
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