CN103409477A - Method for improving saccharic acid conversion rate in L-tryptophan fermentation process - Google Patents
Method for improving saccharic acid conversion rate in L-tryptophan fermentation process Download PDFInfo
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- CN103409477A CN103409477A CN2013103020163A CN201310302016A CN103409477A CN 103409477 A CN103409477 A CN 103409477A CN 2013103020163 A CN2013103020163 A CN 2013103020163A CN 201310302016 A CN201310302016 A CN 201310302016A CN 103409477 A CN103409477 A CN 103409477A
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Abstract
The invention relates to an improved method for preparing L-tryptophan through a fermentation process, and concretely relates to a method for improving the saccharic acid conversion rate in an L-tryptophan fermentation process. In the invention, the content of acetic acid in the fermentation process is substantially reduced by controlling the dissolved oxygen level, the initial glucose concentration, the glucose limited feeding, the specific thallus growth rate and the like according to a cellar metabolic flux distribution regulating principle, so the thallus biomass, the L-tryptophan output and the saccharic acid conversion rate are substantially improved. The method effectively increases the conversion rate in the L-tryptophan fermentation process and substantially improves the L-tryptophan output without adding extra apparatuses or manpower investment, and is suitable for the industrialized production.
Description
Technical field
The present invention relates to a kind of method that improves L-Trp glucose acid invert ratio in the L-Trp fermenting process, belong to the amino acid whose technical field of fermentative Production.
Background technology
L-Trp has another name called tryptophane, chemical name L-Tryptophan, another name L-Tryptophan, L-Tryptophan, molecular formula C
11H
12O
2N
2, relative molecular mass 204.21.L-Trp is one of eight kinds of essential amino acids in human body and animal life activity, and it is present among organism with free state or combined.The growing of humans and animals, metabolism are played an important role, be called as the second indispensable amino acid, be widely used in the aspects such as medicine, food and feed.World market L-Trp annual requirement is about more than 1,200,000 tons, and at present the whole world only has the throughput of more than 10,000 ton, and mainly concentrate on Japan, the countries such as the U.S., China, Korea S, thereby the market outlook of L-Trp are huge.The domestic ground such as Shanghai, Wuhan, Beijing that mainly contain produce the L-Trp for medicine on a small scale on a small quantity, but there is no the L-Trp that the manufacturer production fodder additives is used.Therefore, research raising L-Trp output method has important theory significance and practical value.
Utilize at present the method for microorganisms producing L-Trp to mainly contain enzyme process, microbe transformation method and microbe fermentation method, wherein microbe fermentation method will be the one preferred technique of scale operation L-Trp, and now bacterial strain commonly used has intestinal bacteria, Corynebacterium glutamicum.Because intestinal bacteria have clearer, the easy cultivation of hereditary property, fermentation period is short and can realize the characteristics such as high efficient expression of goal gene, thereby be widely used.But, during Escherichia coli Growth, can produce acetic acid, especially in the high-density culture of recombinant bacterium, the generation of acetic acid and accumulation not only can affect thalli growth, also can suppress the synthetic of product.Therefore, the generation that reduces acetic acid in fermenting process not only is conducive to realize colibacillary high-density culture, and for improving L-Trp output and realizing that the industrialization of L-Trp production is very important.
Summary of the invention
The objective of the invention is to solve intestinal bacteria and can produce the problem of the heteroacid such as acetic acid in the L-Trp fermenting process, provide
A kind of method that improves glucose acid invert ratio in the L-Trp fermenting process.
The inventive methodAdopt measures such as controlling dissolved oxygen level, initial sugar concentration, glucose feeding strategy can significantly improve the L-Trp transformation efficiency, make L-Trp output be significantly improved.
The objective of the invention is to be achieved through the following technical solutions:
The method of glucose acid invert ratio in raising L-Trp fermenting process provided by the present invention, specifically: in the L-Trp fermenting process, adopt the initial sugar concentration of 5 ~ 20g/L, more preferably 10g/L; Control dissolved oxygen level 20 ~ 30%, more preferably 20%; Fermenting process maintains in fermented liquid when glucose concn is 0.05-0.3g/L, glucose max-flow rate of acceleration be 1.6%(per hour in every liter of fermented liquid stream add glucose 16g, lower same), and maintain dissolved oxygen at 20-30% by the speed of stream sugaring, glucose flows rate of acceleration most and should be controlled at as 0.5-1.6%, more preferably 0.8-1.5%.
Principle analysis of the present invention
Optimizing carbon source is the key factor that realizes L-Trp production bacterium high density fermentation, and glucose is current the most frequently used carbon source material.In substratum, the concentration of glucose has a strong impact on the metabolic way of cell, and when sugared concentration surpassed 50 g/L, colibacillary growth will be suppressed.The absorption speed of controlling glucose a little less than or equal the turnover ability of TCA circulation, can effectively reduce the heteroacid such as acetic acid, pyruvic acid, therefore maintaining glucose concn suitable in fermenting process can effectively improve the glucose service efficiency, thereby improves glucose acid invert ratio.
L-Trp 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 affect thalli growth and product and generate.Oxygen has potential toxic side effect to aerobic microbiological, and oxygen concn is too high also can produce the Crabtree effect; Too low oxyty causes a large amount of generations of acetic acid, and thalli growth is suppressed, and specific growth rate descends, and even thalline autolyze has a strong impact on product synthetic.Maintain the oxyty of certain level, not only be conducive to thalli growth, also help the raising acid yield.
Multiple fermentation parameter can reflect the Expenditure Levels of carbon source in fermenting process, as the pH in substratum and DO, and the CO2 concentration in tail gas etc.During the fermentation, in case glucose exhausts, produce the ratio of bacterium and take the photograph sugared rate reduction, specific oxygen utilization rate also decreases, and the DO value is gone up gradually; When taking the photograph sugared speed lower than threshold value, pulse fills into after glucose the ratio that makes to produce bacterium and takes the photograph sugared speed and increase, and specific consumption sugar speed raises, the reduction of DO value.Therefore, pulse fills into the difference that produces signal and can be online adjustment feed rate direct foundation is provided to glucose according to the DO value.Limit during the fermentation the supply of glucose, can reduce particularly organic acid generation of by product.
Advantage of the present invention and beneficial effect:
The present invention proposes and be applicable to improve glucose acid invert ratio method in the L-Trp fermenting process, it is controlled dissolved oxygen level, initial sugar concentration, glucose limitation stream adds and can significantly reduce the heteroacid content in fermenting process by measures such as stream sugaring rate-controlling thalline specific growth rates by employing, and thalline biomass, L-Trp output and glucose acid invert ratio are significantly improved.
The inventive method is in the situation that increase extras and human input, effectively increased the transformation efficiency in the L-Trp fermenting process and significantly improved the output of L-Trp, is suitable for suitability for industrialized production.
Embodiment
The present invention is further illustrated below by embodiment, and the cited case does not limit the scope of the invention:
The embodiment of the present invention 1
The bacterial strain adopted is intestinal bacteria;
Substratum is fermention medium [glucose 20 g/L, yeast extract paste 1 the g/L, (NH generally adopted existing
4)
2SO
44 g/L, Trisodium Citrate 2 g/L, MgSO
47H
2O 5 g/L, KH
2PO
42 g/L, FeSO
47H
2O 100 mg/L];
Cultural method: bacterial classification is accessed to seed culture medium [glucose 10 g/L, yeast extract paste 15 g/L, (NH
4)
2SO
410 g/L, Trisodium Citrate 0.5 g/L, MgSO
47H
2O 5 g/L, KH
2PO
41.5 g/L, FeSO
47H
2O 15 mg/L, V
B1100 mg/L] in, inoculum size is 10%; At 32 ℃, pH, be that 6.8-7.2 and dissolved oxygen are under the 20-30% condition, to cultivate 12-14h to logarithmic phase in the automatic controlled fermentation tank of 5L, inoculum size access by 10% contains in the automatic controlled fermentation tank of 30L of the above fermention medium, initial air flow 2 L/min, mixing speed 500 r/min ~ 800 r/min, dissolved oxygen level is 20-30%, by auto-feeding ammoniacal liquor, control pH at 6.7-7.0,32 ℃ of culture temperature, with bubble enemy froth breaking, and add by stream the glucose solution that concentration is 800g/L, ferment to 35h and stop.Fermention medium initial sugar concentration 10 g/L, maintaining residual sugar mass concentration in fermented liquid is 0.05-0.3g/L, dissolved oxygen 20-30 %, mend sugared speed and be less than 1.6%, fermentation culture 35h, L-Trp are 42.5 g/L, acetic acid content 1.4 g/L, pyruvic acid content 1.1 g/L, phenylalanine content 2.4 g/L, glucose acid invert ratio 18%.
Comparative Examples 1
The bacterial strain adopted is intestinal bacteria; Substratum is the fermention medium (with embodiment 1) generally adopted existing, and glucose concn is 20 g/L; Cultural method is with embodiment 1, and fermentation accumulates L-Trp.Fermentation culture 35h, L-Trp are 37.3 g/L, acetic acid content 4.3 g/L, pyruvic acid content 1.8 g/L, phenylalanine content 3.7 g/L, glucose acid invert ratio 15%.
Comparative Examples 2
The bacterial strain adopted is intestinal bacteria; Substratum and cultural method, with embodiment 1, under the condition of controlling residual sugar 2-5 g/L, maintain the maximum sugared speed 1.6% of mending, fermentation accumulation L-Trp.Fermentation culture 30h, L-Trp are 33.7 g/L, acetic acid content 7.4 g/L, pyruvic acid content 3.1 g/L, phenylalanine content 4.4 g/L, glucose acid invert ratio 10%.
Comparative Examples 3
The bacterial strain adopted is intestinal bacteria; Substratum and cultural method, with embodiment 1, under the condition of controlling dissolved oxygen 25-30%, maintain and mend sugared speed 1.3%, fermentation accumulation L-Trp.Fermentation culture 37h, L-Trp are 35.2 g/L, acetic acid content 1.6 g/L, pyruvic acid content 1.3 g/L, phenylalanine content 2.8 g/L, glucose acid invert ratio 16%.
Comparative Examples 4
The bacterial strain adopted is intestinal bacteria; Substratum and cultural method, with embodiment 1, are controlled dissolved oxygen 30-40%, fermentation accumulation L-Trp.Fermentation culture 33h, L-Trp are 38.5 g/L, acetic acid content 1.8 g/L, pyruvic acid content 2.0 g/L, phenylalanine content 2.9 g/L, glucose acid invert ratio 14%.
The embodiment of the present invention 1 is compared with Comparative Examples 1: in embodiment 1, L-Trp output improves 13.9%, and acetic acid content reduces by 67.4%, and pyruvic acid content reduces by 38.9%, and phenylalanine content reduces by 35.1%, and glucose acid invert ratio improves 20.0%.
The embodiment of the present invention 1 is compared with Comparative Examples 2: in embodiment 1, L-Trp output improves 26.1%, and acetic acid content reduces by 81.1%, and pyruvic acid content reduces by 64.5%, and phenylalanine content reduces by 45.4%, and glucose acid invert ratio improves 80.0%.
The embodiment of the present invention 1 is compared with Comparative Examples 3: in embodiment 1, L-Trp output improves 20.7%, and acetic acid content reduces by 12.5%, and pyruvic acid content reduces by 15.3%, and phenylalanine content reduces by 14.3%, and glucose acid invert ratio improves 12.5%;
The embodiment of the present invention 1 is compared with Comparative Examples 4: in embodiment 1, L-Trp output improves 10.4%, and acetic acid content reduces by 22.2%, and pyruvic acid content reduces by 45.0%, and phenylalanine content reduces by 17.2%, and glucose acid invert ratio improves 28.6%.
Claims (4)
1. a method that improves glucose acid invert ratio in the L-Trp fermenting process, is characterized in that: in the L-Trp fermenting process, adopt 10 ~ 20g/L initial sugar concentration; Control dissolved oxygen level 20 ~ 30%; Fermenting process maintains in fermented liquid when glucose concn is 0.01-0.3 g/L, and glucose max-flow rate of acceleration is 1.6%, and while maintaining dissolved oxygen 20-30%, glucose feeding speed is for being controlled at 0.5-1.6%, and preferably controlling glucose feeding speed is 0.8-1.5%.
2. method according to claim 1, it is characterized in that: in the L-Trp fermenting process, initial sugar concentration is preferably 20g/L.
3. method according to claim 1, is characterized in that: in the L-Trp fermenting process, control dissolved oxygen level preferably 20%.
4. method according to claim 1, is characterized in that: in the L-Trp fermenting process, control glucose preferred streams rate of acceleration 0.8-1.5%.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103627743A (en) * | 2013-12-18 | 2014-03-12 | 江苏江山制药有限公司 | Method for improving fermentation yield of L-tryptophan |
| CN107058417A (en) * | 2016-06-27 | 2017-08-18 | 通辽梅花生物科技有限公司 | A kind of tryptophan upgrading synergy new technology |
| CN107937452A (en) * | 2017-12-07 | 2018-04-20 | 天津科技大学 | A kind of fermentation manufacturing technique of L tryptophans |
| CN108624516A (en) * | 2017-03-20 | 2018-10-09 | 华东理工大学 | A kind of metabolite amount in raising fermented cells and the method for preparing IDMS standard items |
| CN109593801A (en) * | 2018-12-17 | 2019-04-09 | 新疆阜丰生物科技有限公司 | A kind of technique of fermenting and producing L-Trp |
| CN114381477A (en) * | 2020-10-19 | 2022-04-22 | 江苏元易邦生物科技有限公司 | Method for improving yield and sugar-acid conversion rate of L-tryptophan fermentation process |
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| CN101914584A (en) * | 2010-09-03 | 2010-12-15 | 王东阳 | Method for producing L-tryptophan |
| CN101967502A (en) * | 2010-11-02 | 2011-02-09 | 天津科技大学 | Method for reducing content of metabolic byproduct acetic acid in fermentation process of L-tryptophan |
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2013
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| CN101914584A (en) * | 2010-09-03 | 2010-12-15 | 王东阳 | Method for producing L-tryptophan |
| CN101967502A (en) * | 2010-11-02 | 2011-02-09 | 天津科技大学 | Method for reducing content of metabolic byproduct acetic acid in fermentation process of L-tryptophan |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103627743A (en) * | 2013-12-18 | 2014-03-12 | 江苏江山制药有限公司 | Method for improving fermentation yield of L-tryptophan |
| CN103627743B (en) * | 2013-12-18 | 2015-05-20 | 江苏江山制药有限公司 | Method for improving fermentation yield of L-tryptophan |
| CN107058417A (en) * | 2016-06-27 | 2017-08-18 | 通辽梅花生物科技有限公司 | A kind of tryptophan upgrading synergy new technology |
| CN108624516A (en) * | 2017-03-20 | 2018-10-09 | 华东理工大学 | A kind of metabolite amount in raising fermented cells and the method for preparing IDMS standard items |
| CN108624516B (en) * | 2017-03-20 | 2022-08-26 | 华东理工大学 | Method for improving metabolite amount in fermentation cells and preparing IDMS standard substance |
| CN107937452A (en) * | 2017-12-07 | 2018-04-20 | 天津科技大学 | A kind of fermentation manufacturing technique of L tryptophans |
| CN109593801A (en) * | 2018-12-17 | 2019-04-09 | 新疆阜丰生物科技有限公司 | A kind of technique of fermenting and producing L-Trp |
| CN114381477A (en) * | 2020-10-19 | 2022-04-22 | 江苏元易邦生物科技有限公司 | Method for improving yield and sugar-acid conversion rate of L-tryptophan fermentation process |
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Application publication date: 20131127 |