CN101962664A - Fermentation process for producing L-valine efficiently - Google Patents
Fermentation process for producing L-valine efficiently Download PDFInfo
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- CN101962664A CN101962664A CN 201010527435 CN201010527435A CN101962664A CN 101962664 A CN101962664 A CN 101962664A CN 201010527435 CN201010527435 CN 201010527435 CN 201010527435 A CN201010527435 A CN 201010527435A CN 101962664 A CN101962664 A CN 101962664A
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- xie ansuan
- valine
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Abstract
The invention discloses a fermentation process for producing L-valine efficiently, and solves the problems of low acid yield, low sugar-acid conversion rate and the like of the prior production process. In the fermentation process, auxotrophic type and valine leakage type stains are used as producing stains, some nutrients are mixed in a certain ratio to prepare a culture medium with rich and balance nutrients, and the culture medium is used as a fermentation culture medium; and in a fermentation process, dissolved oxygen is kept at a proper level by regulating the stirring speed of a fermentation tank and the air volume, the pH is controlled by adding liquid ammonia, the residual sugar content is kept at a low level by adding glucose solution at a certain concentration, and the fermentation stops 60 hours later. In the invention, extra equipment and human inputs are avoided, the whole fermentation period is shortened, the L-valine yield is improved considerably by over 55g/L, the conversion rate is improved by over 25 percent, the operation in the whole process is simple and the production cost is low. Thus, the process is very suitable for industrial production.
Description
[technical field]: the present invention relates to a kind of zymotechnique of High-efficient Production L-Xie Ansuan, belong to technical field of producing amino acid by fermentation.
[background technology]: L-Xie Ansuan (L-valine) belongs to branched chain amino acid, is one of essential amino acid.In recent years, because amino acid has purposes extremely widely at aspects such as food, medicine, feed, agricultural and daily-use chemical industries, especially the develop rapidly of producing along with anti-cancer drug preparation, amino acid transfusion preparation and sweet taste dipeptides, growing to the amino acid whose demand of raw material, market demand constantly increases.Recently, it is found that the L-Xie Ansuan is the important intermediate of synthetic a kind of immune antibiotic, its year consumption increase sharply to thousands of tons of, world L-Xie Ansuan annual production has now reached more than 10000 tons.In whole amino acid manufacture, along with the continuous growth of amino acid transfusion demand, the demand of L-Xie Ansuan also will increase, and its production occupies an important position.In addition, Xie Ansuan also has a wide range of applications on food, makeup and fodder industry.The Xie Ansuan fermentation is typical metabolic control fermentation.Though the domestic product that natural protein hydrolyzed solution separation and Extraction L-Xie Ansuan is arranged, output is very low, and quality is not good, and purity is not high, can't realize large-scale industrial production.It is low to utilize Production by Microorganism Fermentation L-Xie Ansuan to have a raw materials cost, and reaction conditions is gentle and easily realize advantage such as scale operation, is a kind of very economical production method.But with Production by Microorganism Fermentation L-Xie Ansuan, the acid yield of domestic most of bacterial strains is not high, and particularly the production level of L-Xie Ansuan and output far can not satisfy the demand of domestic market.Therefore, the research of carrying out fermentation production of L-valine is extremely important.
And, along with rising steadily and market competition fierce further of the prices of raw and semifnished materials in recent years, in order when improving Xie Ansuan output and transformation efficiency, to reduce production costs, still have in a large number and report, mainly concentrate on the aspects such as exploitation of mutagenic and breeding Xie Ansuan superior strain, fermenting process control and optimization and new raw material about the research of Xie Ansuan fermentation aspect.
Xie Ansuan is produced Pseudomonas in auxotrophic mutant, and can accumulate Xie Ansuan outside born of the same parents in a large number is because the metabolism of thalline is in error state (ERST), very responsive to envrionment conditions.Fermentation condition such as carbon source, nitrogenous source, inorganic salt, vitamin H, pH, temperature and dissolved oxygen etc. all have material impact to synthesizing of Xie Ansuan.Wherein, substrate (as glucose) concentration is an important parameter of control bacterial metabolism process, the main pathways metabolism of passing through the synthetic or activity influence thalline of control relevant enzyme.Glucose can check, suppress the synthetic of plurality of enzymes by " glucose effect ", and as amino acid synthetase etc., this to check effect mainly be that its catabolite causes.In the Xie Ansuan fermentation that with glucose is carbon source, the catabolite repression of glucose can cause the accumulation of multiple by product such as lactic acid, thereby influences glucose acid invert ratio.In addition, dissolved oxygen also is a vital factor, and the generation of heteroacid and the waste of carbon skeleton and dissolved oxygen concentration are closely related.Therefore must control appropriate oxygen dissolving value during the fermentation,, increase L-Xie Ansuan productive rate, reduce the generation of heteroacid to optimize the metabolism distributions.
At present, China L-Xie Ansuan main batch fermentation pattern that adopts on manufacture.In this production model, it is extremely important for the fermentation of Xie Ansuan to keep certain substrate (as glucose) concentration.For reaching the requirement of economic technology, improve Xie Ansuan output in the single batch of fermenting process and will improve substrate (as glucose) concentration in the initial medium, but concentration of substrate too high meeting causing osmotic pressure is excessive, influences the output of the eubolism and the Xie Ansuan of thalline.Otherwise, the low growth that helps thalline of initial glucose concentration, but the residual sugar in the later stage fermentation liquid exhausts rapidly, and its throughput can not be brought into play to greatest extent.Meanwhile, in present production model, oxyty often can not be well controlled, and it is very big that dissolved oxygen level floats, can not be for a long time stable maintain on the appropriate level.In the Xie Ansuan fermenting process, because the L-Xie Ansuan is synthetic not to be via tricarboxylic acid cycle, but it is synthetic for the prerequisite thing by pyruvic acid, when oxygen supply is not enough, glucose degradation is suppressed by the oxidation of TCA round-robin, pyruvic acid storehouse level improves in the cell, and helping precursor increases, thereby causes L-Xie Ansuan output to increase.And dissolved oxygen is when too high, and the acetolactate synthestase enzyme is lived and obviously reduced, and the TCA circular flow strengthens, and causes the loss of carbon stream; But dissolved oxygen is crossed when hanging down, and TCA cyclic metabolism flow reduces, and is not enough to balance glucose glycolysis speed, thereby has stimulated the enzyme of serum lactic dehydrogenase to live, and the metabolism circulation is generated to lactic acid, causes the lactic acid accumulation.Both of these case is unfavorable for that all Xie Ansuan generates.In addition, existing a lot of L-Xie Ansuan fermentative medium formula all can not well remove to satisfy the growth metabolism of its corresponding production bacterial strain to greatest extent and produce sour requirement.
[summary of the invention]: the objective of the invention is to overcome defectives such as existing Xie Ansuan production method fermentation and acid amount is lower, glucose acid invert ratio is low, a kind of method of efficient fermentative production L-Xie Ansuan is provided.Under the situation that does not increase extras and human input, realized the shortening of whole fermentation period and the significantly raising of L-Xie Ansuan output and transformation efficiency, be suitable for suitability for industrialized production.
The objective of the invention is to be achieved through the following technical solutions: the method for a kind of efficient fermentative production L-Xie Ansuan provided by the invention, comprise seed culture and carry out fermentation and acid, it is characterized in that: in the fermenting process, by regulating fermentor tank mixing speed and air quantity dissolved oxygen is controlled on the proper level, whole fermentation process neither produces the secondary acid of heteroacid such as more lactic acid, do not cause causing carbon source to run off in a large number owing to the TCA circular flow strengthens yet, add liquefied ammonia control pH by stream, add an amount of bubble enemy froth breaking by stream, and residual sugar is controlled at certain level by the glucose solution that stream adds certain higher concentration, make and neither produce the glucose inhibition in the whole fermentation process, also do not constitute substrate restriction, fermenting to 60h stops.
The concrete steps of this method are as follows:
(1) cultivates seed: preparation seed culture medium [glucose 2.5%, yeast powder 0.4%, corn steep liquor 1.5%, soya-bean cake hydrolyzed solution 1%, KH
2PO
43H
2O 0.1%, MgSO
47H
2O 0.04%, MnSO
4H
2O 0.001%, VB
10.00003%, VH 0.00002%, urea 0.05%], 121 ℃ of sterilization 15min; Bacterial classification is inserted in the seed culture medium, and inoculum size is generally 10%; Under suitable temperature, pH and dissolved oxygen condition, control automatically and be cultured to logarithmic phase in the fermentor tank in 5L.
(2) low aerobe fermentation: the kind liquid that step (1) is made is with 10%～20% inoculum size inoculation fermentation, and control fermented liquid temperature is 32 ℃; Feed suitable air, regulate the agitation as appropriate rotating speed, adopting the control of oxygen supply pattern stage by stage dissolved oxygen: 0～18h is about 20%, and 18h is later on 5%～10%; Control pH 6.7～7.0 by the liquefied ammonia of auto-feeding 25%; Add an amount of bubble enemy froth breaking by stream; And to add concentration by stream be that the glucose solution of 600～900g/L is controlled at 0.5～2.0% with residual sugar, ferments to 60h to stop.
Advantage of the present invention and positively effect:
Compare with existing technology, method of the present invention has following characteristics:
1, the present invention's glucose solution of adding higher concentration by proper flow is controlled at appropriate level with remaining sugar concentration, make and cause because of glucose concn is too high neither in the whole fermentation process that osmotic pressure is excessive and produce " glucose effect " and check catabolite, do not cross the low substrate restriction that constitutes because of glucose concn yet, residual sugar in the fermented liquid is exhausted rapidly, cause bacterial classification throughput not brought into play to greatest extent;
2, the present invention is controlled at oxyty on the appropriate level by suitable adjusting fermentor tank mixing speed and air quantity, making in the whole fermentation process neither causes TCA cyclic metabolism flow to reduce because of oxyty is low excessively, be not enough to balance glucose glycolysis speed, thereby stimulated the enzyme of serum lactic dehydrogenase to live, the metabolism circulation is generated to lactic acid, cause the lactic acid accumulation,, the too high TCA of the causing circular flow of oxyty do not generate a large amount of losses that a large amount of CO2 cause carbon source because of strengthening yet, and cause the obviously reduction alive of acetolactate synthestase enzyme, cause L-Xie Ansuan output to descend;
3, the present invention is comparing under the situation that does not increase any extras and human input with existing production technique, shortened whole fermentation period, seed culture foreshortens to 7～9h, and the output (more than the 55g/L) and the transformation efficiency (more than 25%) of L-Xie Ansuan have significantly been improved, whole simple operation of process, production cost is lower, and remarkable in economical benefits very is suitable for suitability for industrialized production.
[embodiment]:
Embodiment 1:
The microbial strains of using produces bacterium for existing Xie Ansuan.Efficiently ferment with above-mentioned bacterial strains, used substratum has two kinds:
(1) seed culture medium: glucose 2.5%, yeast powder 0.4%, corn steep liquor 1.5%, soya-bean cake hydrolyzed solution 1%, KH
2PO
40.1%, MgSO
47H
2O 0.04%, MnSO
4H
2O 0.001%, VB
1300ug/L, VH 200ug/L, urea 0.05%.Transfer pH to 6.7～7.0,121 ℃ sterilization 15min with NaOH and hydrochloric acid.
(2) fermention medium: glucose 6%, corn steep liquor 2.5%, soya-bean cake hydrolyzed solution 2.5%, KH
2PO
40.05%, MgSO
47H
2O 0.02%, MnSO
4H
2O 0.001%, Met 0.05%, Ile 0.006%, Leu 0.02%, V
B10.00001%, VH 0.000008%.Transfer pH to 6.7～7.0,115 ℃ sterilization 20min with NaOH and hydrochloric acid.
Bacterial classification is inserted in the seed culture medium, and inoculum size is 10%; At 32 ℃, pH be 7.0 and dissolved oxygen be to control automatically in the fermentor tank in 5L under 20% condition to cultivate 8h to logarithmic phase, inoculum size by 10% inserts the 5L that contains fermention medium and controls in the fermentor tank automatically, control fermented liquid temperature is 32 ℃, feed suitable air, regulate the agitation as appropriate rotating speed, adopting the control of oxygen supply pattern stage by stage dissolved oxygen: 0～18h is 20%, 18～60h is 5%, control pH 6.7～7.0 by auto-feeding liquefied ammonia, add an amount of bubble enemy froth breaking by stream, and to add concentration by stream be that the glucose solution of 600g/L is controlled at 0.5% with residual sugar, ferments to 60h to stop.The output of L-Xie Ansuan is 56g/L, and glucose acid invert ratio is 25.5%.
Embodiment 2:
The microbial strains of using produces bacterium for existing Xie Ansuan.Efficiently ferment with above-mentioned bacterial strains, used substratum has two kinds:
(1) seed culture medium: with embodiment 1.
(2) fermention medium: glucose 10%, corn steep liquor 1.5%, soya-bean cake hydrolyzed solution 3.5%, KH
2PO
40.1%, MgSO
47H
2O 0.03%, MnSO
40.001%, Met 0.05%, Ile 0.006%, Leu0.02%, V
B10.00002%, VH 0.000015%.Transfer pH to 6.7～7.0,115 ℃ sterilization 20min with NaOH and hydrochloric acid.
Bacterial classification is inserted in the seed culture medium, and inoculum size is 10%; At 32 ℃, pH be 7.0 and dissolved oxygen be to control automatically in the fermentor tank in 5L under 20% condition to cultivate 8h to logarithmic phase, inoculum size by 10% inserts the 5L that contains fermention medium and controls in the fermentor tank automatically, control fermented liquid temperature is 32 ℃, feed suitable air, regulate the agitation as appropriate rotating speed, adopting the control of oxygen supply pattern stage by stage dissolved oxygen: 0～18h is 20%, 18～60h is 5%, control pH 6.7～7.0 by auto-feeding liquefied ammonia, add an amount of bubble enemy froth breaking by stream, and to add concentration by stream be that the glucose solution of 800g/L is controlled at residual sugar about 2%, ferments to 60h to stop.The output of L-Xie Ansuan is 57g/L, and glucose acid invert ratio is 26%.
Embodiment 3:
The microbial strains of using produces bacterium for existing Xie Ansuan.Efficiently ferment with above-mentioned bacterial strains, used substratum has two kinds:
(1) seed culture medium: with embodiment 1.
(2) fermention medium: glucose 8%, corn steep liquor 0.5%, soya-bean cake hydrolyzed solution 4.5%, KH
2PO
40.1%, MgSO
47H
2O 0.04%, MnSO
40.001%, Met 0.05%, Ile 0.006%, Leu0.02%, V
B10.00002%, VH 0.000015%.Transfer pH to 6.7～7.0,115 ℃ sterilization 20min with NaOH and hydrochloric acid.
Bacterial classification is inserted in the seed culture medium, and inoculum size is 10%; At 32 ℃, pH be 7.0 and dissolved oxygen be to control automatically in the fermentor tank in 5L under 20% condition to cultivate 8h to logarithmic phase, inoculum size by 10% inserts the 5L that contains fermention medium and controls in the fermentor tank automatically, control fermented liquid temperature is 32 ℃, feed suitable air, regulate the agitation as appropriate rotating speed, adopting the control of oxygen supply pattern stage by stage dissolved oxygen: 0～18h is 20%, 18～60h is 5%, control pH 6.7～7.0 by auto-feeding liquefied ammonia, add an amount of bubble enemy froth breaking by stream, and to add concentration by stream be that the glucose solution of 900g/L is controlled at 1～1.5% with residual sugar, ferments to 60h to stop.The output of L-Xie Ansuan is 59g/L, and glucose acid invert ratio is 27%.
Claims (4)
1. the zymotechnique of a High-efficient Production L-Xie Ansuan, comprise seed culture and carry out hypoxemia control fermentation, it is characterized in that: in the fermenting process, by regulating fermentor tank mixing speed and air quantity dissolved oxygen is controlled on the proper level, make whole fermentation process neither produce more lactic acid, also do not cause generating a large amount of CO because the TCA circular flow strengthens
2And cause carbon source to run off in a large number, and add liquefied ammonia control pH by stream, add an amount of bubble enemy froth breaking by stream, and add certain density glucose solution by stream residual sugar is controlled at lower level, make neither to produce glucose in the whole fermentation process and suppress, also do not constitute the substrate restriction, fermenting to 60h stops.
2. method according to claim 1 is characterized in that: stream sugaring concentration is 600～900g/L in the fermenting process, and residual sugar is controlled at 0.5～2.0%.
3. method according to claim 1 is characterized in that: described dissolved oxygen of fermentation liquid concentration is control stage by stage: 0～18h is 10-30%, and 18h is later on 5-15%.
4. method according to claim 1 is characterized in that: the per-cent of each ingredients constitute total amount is in the described fermented liquid: inoculum size 10～20%, glucose 6～10%, corn steep liquor 0.5～2.5%, soya-bean cake hydrolyzed solution 2.5～4.5%, MgSO
47H
2O 0.02～0.04%, MnSO
4H
2O 0.0005～0.001%, Met0.05%, Ile 0.006%, Leu 0.02%, V
B10.00001～0.00002%, VH0.000008%～0.000015%.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695526A (en) * | 2016-04-06 | 2016-06-22 | 河南巨龙生物工程股份有限公司 | Fermentation technique for enhancing L-valine acid productivity by multi-step material supplementation |
| CN106086099A (en) * | 2016-08-27 | 2016-11-09 | 河北圣雪大成制药有限责任公司 | L valine fermentation medium and the fermentation process with its production L valine |
| CN109652476A (en) * | 2019-02-25 | 2019-04-19 | 内蒙古拜克生物有限公司 | A kind of method of fermenting and producing Valine |
| CN111172086A (en) * | 2020-03-18 | 2020-05-19 | 通辽梅花生物科技有限公司 | Fermentation method for producing L-isoleucine |
| CN112626143A (en) * | 2020-12-14 | 2021-04-09 | 天津科技大学 | Fermentation method of L-lysine |
| CN114277064A (en) * | 2021-12-07 | 2022-04-05 | 河南星汉生物科技有限公司 | Production process for preparing highlight pure L-lactic acid from straw |
| CN114438145A (en) * | 2022-03-30 | 2022-05-06 | 通辽梅花生物科技有限公司 | Method for producing L-valine by fermentation |
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| US6737255B2 (en) * | 2000-01-26 | 2004-05-18 | Ajinomoto Co., Inc. | Mutant ilvH gene and method for producing L-valine |
| CN1982466A (en) * | 2005-12-12 | 2007-06-20 | 上海化工研究院 | Production of stabilized isotope 15N labelled L-valine |
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2010
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| US6737255B2 (en) * | 2000-01-26 | 2004-05-18 | Ajinomoto Co., Inc. | Mutant ilvH gene and method for producing L-valine |
| CN1982466A (en) * | 2005-12-12 | 2007-06-20 | 上海化工研究院 | Production of stabilized isotope 15N labelled L-valine |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695526A (en) * | 2016-04-06 | 2016-06-22 | 河南巨龙生物工程股份有限公司 | Fermentation technique for enhancing L-valine acid productivity by multi-step material supplementation |
| CN106086099A (en) * | 2016-08-27 | 2016-11-09 | 河北圣雪大成制药有限责任公司 | L valine fermentation medium and the fermentation process with its production L valine |
| CN109652476A (en) * | 2019-02-25 | 2019-04-19 | 内蒙古拜克生物有限公司 | A kind of method of fermenting and producing Valine |
| CN111172086A (en) * | 2020-03-18 | 2020-05-19 | 通辽梅花生物科技有限公司 | Fermentation method for producing L-isoleucine |
| CN112626143A (en) * | 2020-12-14 | 2021-04-09 | 天津科技大学 | Fermentation method of L-lysine |
| CN112626143B (en) * | 2020-12-14 | 2022-11-25 | 天津科技大学 | Fermentation method of L-lysine |
| CN114277064A (en) * | 2021-12-07 | 2022-04-05 | 河南星汉生物科技有限公司 | Production process for preparing highlight pure L-lactic acid from straw |
| CN114438145A (en) * | 2022-03-30 | 2022-05-06 | 通辽梅花生物科技有限公司 | Method for producing L-valine by fermentation |
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Application publication date: 20110202 |