CN101967502A - Method for reducing content of metabolic byproduct acetic acid in fermentation process of L-tryptophan - Google Patents
Method for reducing content of metabolic byproduct acetic acid in fermentation process of L-tryptophan Download PDFInfo
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- CN101967502A CN101967502A CN 201010527463 CN201010527463A CN101967502A CN 101967502 A CN101967502 A CN 101967502A CN 201010527463 CN201010527463 CN 201010527463 CN 201010527463 A CN201010527463 A CN 201010527463A CN 101967502 A CN101967502 A CN 101967502A
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- tryptophane
- acetic acid
- tryptophan
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Abstract
The invention provides a method for reducing the content of metabolic byproduct acetic acid in the fermentation process of L-tryptophan. The invention relates to an improved fermentation method for preparing L-tryptophan. According to the principle of adjusting cellular metabolic flow distribution, by adopting the measures of controlling the level of dissolved oxygen, the initial concentration of sugar, the limited flow of glucose, the specific growth rate of thalli and the like, the invention obviously reduces the content of acetic acid in the fermentation process, thereby obviously improving the biomass of the thalli and the yield of the L-tryptophan.
Description
[technical field]: the present invention relates to the method for metabolic by-prods acetate in a kind of L-of minimizing tryptophane fermenting process, belong to technical field of producing amino acid by fermentation.
[background technology]: the L-tryptophane has another name called tryptophane, chemical name L-2-amino-3-indyl propionic acid, another name L-pancreas Argine Monohydrochloride, L-amino indole propionic acid, molecular formula C
11H
12O
2N
2, relative molecular mass 204.21.The L-tryptophane is one of eight kinds of essential amino acids in human body and the animal life activity, and it is present among the organism with free state or combined.The growing of humans and animals, metabolism are played an important role, be called as second indispensable amino acid, be widely used in aspects such as medicine, food and feed.World market L-tryptophane annual requirement is about more than 1,200,000 tons, and at present the whole world has only more than 10,000 ton throughput, and mainly concentrate on Japan, countries such as the U.S., China, Korea S, thereby the market outlook of L-tryptophane are huge.The domestic ground small-scale produced in small quantities such as Shanghai, Wuhan, Beijing that mainly contain are used for the L-tryptophane of medicine, but still do not have the L-tryptophane that the manufacturer production fodder additives is used.Therefore, research raising L-tryptophane output method has important significance for theories and practical value.
Utilize the method for microorganisms producing L-tryptophane to mainly contain enzyme process, microbe transformation method and microbe fermentation method at present, wherein microbe fermentation method will be the one preferred technique of scale operation L-tryptophane, and bacterial strain now commonly used has intestinal bacteria, Corynebacterium glutamicum.Because intestinal bacteria have clearer, the easy cultivation of hereditary property, the fermentation period weak point also can be realized characteristics such as efficiently expressing of goal gene, thereby are used widely.But intestinal bacteria can produce acetate during growth, and especially in the high-density culture of reorganization bacterium, the generation of acetate and accumulation not only can influence thalli growth, also can suppress the synthetic of product.Therefore, the generation that reduces acetate in the fermenting process not only helps realizing colibacillary high-density culture, and for improving L-tryptophane output and realizing that the industrialization of L-tryptophane production is very important.
[summary of the invention]: the present invention can produce acetate according to intestinal bacteria in L-tryptophane fermenting process, suppress thalli growth, causes L-tryptophane output to reduce.Measures such as employing control dissolved oxygen level, first sugared concentration, glucose limitation stream add can significantly reduce the acetic acid content in the fermenting process, make L-tryptophane output be significantly improved.
During the fermentation, the acetate generation mainly contains two reasons, i.e. the breathing of the not enough feasible reorganization bacterium of the oxygen delivery capacity of equipment is restricted with the absorption speed of glucose has enough to meet the need ability greater than reorganization bacterium TCA round-robin.Both of these case all can cause substrate to be converted into acetate by the acetylphosphate approach, and the metabolism load that exogenous gene expression produces can reduce colibacillary aerobic repiration ability, easier accumulation acetate in the bacteria growing process that causes recombinating.
The L-tryptophane is produced bacterium in process of growth, needs a large amount of oxygen to participate in metabolism, and oxyty is too high or too low, all can influence thalli growth and product and generate.Oxygen has the potential toxic side effect to aerobic microbiological, and oxygen concn is too high also can to produce the Crabtree effect; Cross low oxyty and then cause a large amount of generations of acetate, thalli growth is suppressed, and specific growth rate descends, even the thalline autolyze, and it is synthetic to have a strong impact on product.Keep the oxyty of certain level, not only help thalli growth, also help the raising acid yield.
Optimizing carbon source is the key factor that realizes L-tryptophane production bacterium high density fermentation, and glucose is present the most frequently used carbon source material.The concentration of glucose has a strong impact on the metabolic way of cell in the substratum, and when sugared concentration surpassed 50g/L, colibacillary growth will be suppressed.When the ratio of producing bacterium when the L-tryptophane is taken the photograph sugared speed and is surpassed certain threshold value, even E.coli also the Crabtree effect can take place and produce acetate under the oxygen sufficiency.The absorption speed of control glucose a little less than or equal TCA round-robin turnover ability, can effectively avoid the intermediate product acetyl-CoA to produce acetate by the acetylphosphate approach, therefore keep glucose concn suitable in the fermenting process and can effectively reduce acetate and produce.
This method effectively reduces the generation of acetate in the L-tryptophane fermenting process and has significantly improved the output of L-tryptophane under the situation that does not increase extras and human input, is suitable for suitability for industrialized production.
The objective of the invention is to be achieved through the following technical solutions:
The invention provides the method that reduces metabolic by-prods acetate in the L-tryptophane fermenting process, it is characterized in that: in L-tryptophane fermenting process, adopt 10～20g/L sugared concentration just, more preferably 20g/L, the control dissolved oxygen level is 20～30%, more preferably 20%, keep that glucose concn is 2～5g/L, more preferably 2g/L in the fermented liquid.
The present invention proposes the method that reduces by-product acetic acid in the L-tryptophane fermenting process that is applicable to, it by adopt control dissolved oxygen level, first sugared concentration, glucose limitation stream adds and control measure such as thalline specific growth rate and can significantly reduce acetic acid content in the fermenting process, and thalline biomass and L-tryptophane output are significantly improved.
[embodiment]:
The present invention is further illustrated below by embodiment, and the cited case does not limit protection scope of the present invention:
Embodiment 1:
The bacterial strain that adopts is intestinal bacteria; Substratum is fermention medium [glucose 20g/L, yeast extract paste the 1g/L, (NH that generally adopts existing
4)
2SO
44g/L, Trisodium Citrate 2g/L, MgSO
47H
2O 5g/L, KH
2PO
42g/L, FeSO
47H
2O 100mg/L]; Cultural method: bacterial classification is inserted seed culture medium [glucose 20g/L, yeast extract paste 15g/L, (NH
4)
2SO
410g/L, Trisodium Citrate 0.5g/L, MgSO
47H
2O5g/L, KH
2PO
41.5g/L, FeSO
47H
2O 15mg/L, V
B1100mg/L] in, inoculum size is 10%; 32 ℃, pH be 7.0 and dissolved oxygen be to control automatically in 5L under 20% condition to cultivate 12h in the fermentor tank to logarithmic phase, inoculum size by 10% inserts the 30L that contains fermention medium and controls in the fermentor tank automatically, initial air flow 2L/min, mixing speed 500r/min～800r/min, dissolved oxygen level is 10%, control pH 7.0 by auto-feeding ammoniacal liquor, 32 ℃ of culture temperature, with bubble enemy froth breaking, and to add concentration by stream be that the glucose solution of 800g/L is controlled at 2g/L with residual sugar, ferments to 40h to stop.When putting jar, the metabolic by-prods acetic acid content is 0.9g/L, and L-tryptophane output is 32.8g/L, and comparison tests according to the facts that (the metabolic by-prods acetic acid content is 2.9g/L, L-tryptophane output is 23.4g/L) acetic acid content reduced 68.96%, and L-tryptophane output has improved 40.17%.
Embodiment 2:
The bacterial strain that adopts is intestinal bacteria; Substratum is for being 10g/L at existing fermention medium (with the embodiment 1) glucose concn that generally adopts; Cultural method is with embodiment 1.When putting jar, the metabolic by-prods acetic acid content is 1.3g/L, and L-tryptophane output is 30.2g/L, and comparison tests according to the facts that (the metabolic by-prods acetic acid content is 2.9g/L, L-tryptophane output is 23.4g/L) acetic acid content reduced 55.17, and L-tryptophane output has improved 29.06%.
Embodiment 3:
The bacterial strain that adopts is intestinal bacteria; Substratum is the fermention medium (with embodiment 1) that generally adopts existing; Cultural method is with embodiment 1, and the control dissolved oxygen level is 30%.When putting jar, the metabolic by-prods acetic acid content is 1.5, and L-tryptophane output is 28.7g/L, and comparison tests according to the facts that (the metabolic by-prods acetic acid content is 2.9g/L, L-tryptophane output is 23.4g/L) acetic acid content reduced 48.27%, and L-tryptophane output has improved 22.65%.
Embodiment 4:
The bacterial strain that adopts is intestinal bacteria; Substratum is the fermention medium (with embodiment 1) that generally adopts existing; Cultural method is with embodiment 1, and adding concentration by stream is that the glucose solution of 800g/L is controlled at 5g/L with residual sugar.When putting jar, the metabolic by-prods acetic acid content is 1.6, and L-tryptophane output is 26.71g/L, and comparison tests according to the facts that (the metabolic by-prods acetic acid content is 2.9g/L, L-tryptophane output is 23.4g/L) acetic acid content reduced 44.83%, and L-tryptophane output has improved 14.14%.
Claims (4)
1. the invention provides the method that reduces metabolic by-prods acetate in the L-tryptophane fermenting process, it is characterized in that: in L-tryptophane fermenting process, adopt 10～20g/L sugared concentration just, the control dissolved oxygen level is 20～30%, keeps that glucose concn is 2～5g/L in the fermented liquid.
2. method according to claim 1 is characterized in that: in L-tryptophane fermenting process, first sugared concentration is 10～20g/L.
3. method according to claim 1 is characterized in that: in L-tryptophane fermenting process, the control dissolved oxygen level is 20～30%.
4. method according to claim 1 is characterized in that: in L-tryptophane fermenting process, keep that glucose concn is 2～5g/L in the fermented liquid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409477A (en) * | 2013-07-18 | 2013-11-27 | 天津科技大学 | Method for improving saccharic acid conversion rate in L-tryptophan fermentation process |
| CN108660168A (en) * | 2017-03-27 | 2018-10-16 | 上海医药工业研究院 | A kind of zymotechnique improving L-Trp yield |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101323867A (en) * | 2008-03-14 | 2008-12-17 | 江苏诚意药业有限公司 | Fermentation process for industrialized production of L-tryptophan by biofermentation method |
-
2010
- 2010-11-02 CN CN 201010527463 patent/CN101967502A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101323867A (en) * | 2008-03-14 | 2008-12-17 | 江苏诚意药业有限公司 | Fermentation process for industrialized production of L-tryptophan by biofermentation method |
Non-Patent Citations (2)
| Title |
|---|
| 《微生物学通报》 20100220 程立坤等 代谢副产物乙酸对L-色氨酸发酵的影响 166-173 第37卷, 第2期 2 * |
| 《食品与发酵工业》 20100330 程立坤等 葡萄糖浓度对大肠杆菌发酵L-色氨酸的影响 5-9 第36卷, 第3期 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409477A (en) * | 2013-07-18 | 2013-11-27 | 天津科技大学 | Method for improving saccharic acid conversion rate in L-tryptophan fermentation process |
| CN108660168A (en) * | 2017-03-27 | 2018-10-16 | 上海医药工业研究院 | A kind of zymotechnique improving L-Trp yield |
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Application publication date: 20110209 |