CN101962663A - High-efficiency fermenting method for producing L-isoleucine - Google Patents
High-efficiency fermenting method for producing L-isoleucine Download PDFInfo
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- CN101962663A CN101962663A CN 201010527464 CN201010527464A CN101962663A CN 101962663 A CN101962663 A CN 101962663A CN 201010527464 CN201010527464 CN 201010527464 CN 201010527464 A CN201010527464 A CN 201010527464A CN 101962663 A CN101962663 A CN 101962663A
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- isoleucine
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- dissolved oxygen
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- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 title claims abstract description 44
- 229960000310 isoleucine Drugs 0.000 title claims abstract description 44
- 229930182844 L-isoleucine Natural products 0.000 title claims abstract description 25
- 238000004519 manufacturing process Methods 0.000 title abstract description 17
- 238000000855 fermentation Methods 0.000 claims abstract description 35
- 230000004151 fermentation Effects 0.000 claims abstract description 35
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 33
- 239000008103 glucose Substances 0.000 claims abstract description 33
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 25
- 239000001301 oxygen Substances 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 22
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 13
- 230000008569 process Effects 0.000 claims abstract description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 14
- 239000002054 inoculum Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 7
- 239000004310 lactic acid Substances 0.000 claims description 7
- 235000014655 lactic acid Nutrition 0.000 claims description 7
- 238000011218 seed culture Methods 0.000 claims description 7
- 239000000758 substrate Substances 0.000 claims description 7
- 244000068988 Glycine max Species 0.000 claims description 6
- 235000010469 Glycine max Nutrition 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 229910021529 ammonia Inorganic materials 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- 238000012262 fermentative production Methods 0.000 claims description 5
- 238000010564 aerobic fermentation Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 239000004615 ingredient Substances 0.000 claims 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 abstract description 19
- 239000002253 acid Substances 0.000 abstract description 8
- 238000009776 industrial production Methods 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 abstract 2
- 235000012041 food component Nutrition 0.000 abstract 1
- 235000015097 nutrients Nutrition 0.000 abstract 1
- 238000003756 stirring Methods 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 230000004060 metabolic process Effects 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 235000011130 ammonium sulphate Nutrition 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 238000009825 accumulation Methods 0.000 description 4
- 238000013019 agitation Methods 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 230000011218 segmentation Effects 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 150000005693 branched-chain amino acids Chemical class 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 230000034659 glycolysis Effects 0.000 description 2
- 230000012666 negative regulation of transcription by glucose Effects 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 229930003756 Vitamin B7 Natural products 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 230000037358 bacterial metabolism Effects 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
一种高效发酵生产L-异亮氨酸的方法。解决现有生产工艺产酸量较低且糖酸转化率低等问题。本发明包括：利用本实验室产异亮氨酸的菌株，以一些营养组分按一定比例配制成营养丰富且均衡的培养基作为发酵培养基，发酵过程中，通过调节发酵罐搅拌转速和风量将溶氧控制在适当的水平上，通过流加液氨控制pH，并通过流加一定浓度的葡萄糖溶液将残糖控制在较低水平，发酵至60h停止。本发明在不增加任何额外设备和人力投入的情况下，缩短了整个发酵周期，并大幅提高了L-异亮氨酸的产量(28g/L以上)和转化率(18％以上)，整个工艺过程操作简便，生产成本较低，十分适合于工业化生产。A method for efficiently producing L-isoleucine by fermentation. The problem of low acid production and low sugar-acid conversion rate in the existing production process is solved. The invention includes: using the strains producing isoleucine in this laboratory, formulating a nutrient-rich and balanced medium with some nutritional components in a certain proportion as a fermentation medium; during the fermentation process, by adjusting the stirring speed and air volume Control the dissolved oxygen at an appropriate level, control the pH by feeding liquid ammonia, and control the residual sugar at a lower level by feeding a certain concentration of glucose solution, and stop the fermentation until 60h. The present invention shortens the entire fermentation period without adding any additional equipment and manpower input, and greatly increases the output (above 28g/L) and conversion rate (above 18%) of L-isoleucine. The process is easy to operate, the production cost is low, and it is very suitable for industrial production.
Description
[technical field]: the present invention relates to a kind of method of efficient fermentative production L-Isoleucine, belong to technical field of producing amino acid by fermentation.
[background technology]: the L-Isoleucine is one of eight kinds of indispensable amino acids of human body, is again one of three kinds of branched chain amino acids simultaneously, can treat neurological disorder, appetite stimulator and anaemia, occupies the status of particularly important in human life's metabolism.But because at present the L-Isoleucine costs an arm and a leg, therefore be mainly used in the preparation mixed amino acid, be applied to high branched-chain amino acid transfusion (as the 3H transfusion etc.) and oral liquid (liver peace dry syrup etc.) especially.So Isoleucine is all having a wide range of applications aspect medicine, the food.Japan adds up to annual production 400-500 ton accounting for monopoly position aspect the production L-Isoleucine in the world at present.In view of the L-Isoleucine produce highly difficult, the L-Isoleucine is a high price amino acid always, world market pharmaceutical grade price is up to 60-80 dollar/kg.Domestic because bacterial classification produces sour poor performance, technology falls behind, and the L-Isoleucine yields poorly, and the overwhelming majority relies on import, especially pharmaceutical grade Isoleucine, consume a large amount of foreign exchanges every year.Compare with Japan, the L-Isoleucine production level of China is also very low, and the throughput of Isoleucine is also very unbecoming with China's great demand.Isoleucine fermentation is typical metabolic control fermentation, and L-Isoleucine production method has extraction method, chemical synthesis and fermentation method three classes, but on industrial production, implement at present have only fermentation method.Because L-Isoleucine and other isomer separation difficulty that chemical synthesis is produced, thereby fail to realize suitability for industrialized production.Fermentation method is exactly a metabolism of utilizing microorganism, and biosynthesizing and excess accumulation L-Isoleucine comprise and add precursor fermentation method and direct fermentation two classes.At present, that is that all right is ripe for the production technology of isoleucine fermentation.People are doing effort aspect raising Isoleucine output and transformation efficiency, mainly concentrate on aspects such as genetic engineering means directive breeding Isoleucine superior strain, fermenting process control and optimization.
In the isoleucine fermentation process, fermentation condition such as vitamin H, carbon source, nitrogenous source, inorganic salt, pH, temperature and dissolved oxygen etc. all have material impact to synthesizing of Isoleucine.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.Be in the isoleucine fermentation of carbon source with glucose, 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 suitable dissolved oxygen during the fermentation,, increase L-Isoleucine productive rate, reduce the generation of heteroacid to change the metabolism distributions.
At present, China's isoleucine fermentation industry mainly adopts the fed-batch fermentation pattern.In this production model, it is extremely important for the fermentation of Isoleucine to keep certain substrate (as glucose) concentration.For reaching the requirement of economic technology, improve glutamic acid yield in the single batch of fermenting process and will improve substrate (as glucose) concentration in the initial medium, but substrate (as glucose) excessive concentration causes that osmotic pressure is excessive, influences the output of the eubolism and the Isoleucine 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 is not that the oxygen supply deficiency is exactly that oxygen supply is excessive, and dissolved oxygen level float very big, can not be for a long time stable maintain on the appropriate level.Under the low excessively condition of dissolved oxygen, 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; And dissolved oxygen is when too high, and the TCA circular flow strengthens, and generates a large amount of CO
2, causing the carbon source loss, two kinds of situations are unfavorable for that all Isoleucine generates.In addition, existing a lot of L-isoleucine fermentation culture medium prescription 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 Isoleucine production method fermentation and acid amount is lower, glucose acid invert ratio is low, a kind of method of efficient fermentative production L-Isoleucine is provided.Under the situation that does not increase extras and human input, the shortening of whole fermentation period and the raising of L-Isoleucine output and transformation efficiency have been realized.
The objective of the invention is to be achieved through the following technical solutions:
The method of a kind of efficient fermentative production L-Isoleucine provided by the invention, comprise seed culture and carry out aerobic 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 certain level, make neither to produce glucose in the whole fermentation process and suppress, also do not constitute the substrate restriction, fermenting to 60h stops.
The concrete steps of this method are as follows:
(1) cultivates seed: preparation seed culture medium [glucose 3%, yeast powder 0.5%, ammonium sulfate 0.5% soya-bean cake hydrolyzed solution 2.5%, corn steep liquor 3%, KH
2PO
43H
2O 0.2%, MgSO
47H
2O 0.08%], 121 ℃ of sterilization 15min; Bacterial classification is inserted in the seed culture medium, and inoculum size is generally 5%; Under suitable temperature, pH and dissolved oxygen condition, control automatically and be cultured to logarithmic phase in the fermentor tank in 5L.
(2) aerobic fermentation: the kind liquid that step (1) is made is with 10%～20% inoculum size inoculation fermentation, and segmentation control fermented liquid temperature: 0～45h is 31 ℃, and 46～60h is 28 ℃; Feed suitable air, regulate the agitation as appropriate rotating speed, adopting the control of oxygen supply pattern stage by stage dissolved oxygen: 0～20h is that 25%, 20～60h is 15%; Control pH 7.0～7.2 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 500～800g/L is controlled at 0.8～2.0% 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, also do not generate a large amount of losses that a large amount of CO2 cause carbon source because of the too high TCA of the causing circular flow of oxyty strengthens;
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, fermentation period is cultivated and is foreshortened to 56～60h, and the output (more than the 28g/L) and the transformation efficiency (more than 18%) of L-Isoleucine 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 Isoleucine.Bacterial classification is inserted seed culture medium [glucose 3%, yeast powder 0.5%, ammonium sulfate 0.5%, soya-bean cake hydrolyzed solution 2.5%, corn steep liquor 3%, KH
2PO
43H
2O 0.2%, MgSO
47H
2O 0.08%.Transfer pH to 7.0～7.2,121 ℃ sterilization 15min with NaOH and hydrochloric acid] in, inoculum size is 5%; 31 ℃, pH be 7.0 and dissolved oxygen be to control automatically in 5L under 20% condition to cultivate 15h in the fermentor tank to the logarithmic growth middle and later periods, the inoculum size by 10% inserts and contains fermention medium [glucose 8%, ammonium sulfate 0.4%, corn steep liquor 1%, soya-bean cake hydrolyzed solution 3%, MgSO
47H
2O 0.1%, FeSO
47H
2O 0.002%, MnSO
4H
2O 0.002%, KH
2PO
40.1%, V
B10.00001%, V
H0.00001%.Transfer pH to 7.0～7.2 with NaOH and hydrochloric acid, 115 ℃ the sterilization 15min] 5L control in the fermentor tank automatically, segmentation control fermented liquid temperature 0～45h is 31 ℃, 46～60h is 28 ℃, feed suitable air, regulate the agitation as appropriate rotating speed, adopting the control of oxygen supply pattern stage by stage dissolved oxygen: 0～20h is 25%, 20～60h is 15%, control pH 7.0～7.2 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 500g/L is controlled at 0.8% with residual sugar, fermenting to 60h stops.The output of L-Isoleucine is 28.9g/L, and glucose acid invert ratio is 18.3%.
Embodiment 2:
The microbial strains of using produces bacterium for existing Isoleucine.Bacterial classification is inserted in the seed culture medium (with embodiment 1), and inoculum size is 5%; 31 ℃, pH be 7.0 and dissolved oxygen be to control automatically in 5L under 20% condition to cultivate 15h in the fermentor tank to the logarithmic growth middle and later periods, the inoculum size by 15% inserts and contains fermention medium [glucose 10%, ammonium sulfate 0.6%, corn steep liquor 1.2%, soya-bean cake hydrolyzed solution 3.3%, MgSO
47H
2O 0.15%, FeSO
47H
2O 0.0025%, MnSO
4H
2O 0.0025%, KH
2PO
40.15%, V
B10.000015%, V
H0.000015%.Transfer pH to 7.0～7.2 with NaOH and hydrochloric acid, 115 ℃ the sterilization 15min] 5L control in the fermentor tank automatically, 31 ℃ of control fermented liquid temperature feed suitable air, regulate the agitation as appropriate rotating speed, adopting the control of oxygen supply pattern stage by stage dissolved oxygen: 0～20h is 25%, 20～60h is 15%, 7.0～7.2, adds an amount of bubble enemy froth breaking by stream by auto-feeding liquefied ammonia control pH, and to add concentration by stream be that the glucose solution of 800g/L is controlled at 0.8～2.0% with residual sugar, ferments to 60h to stop.The output of L-Isoleucine is 29.6g/L, and glucose acid invert ratio is 19.4%.
Embodiment 3:
The microbial strains of using produces bacterium for existing Isoleucine.Bacterial classification is inserted in the seed culture medium (with embodiment 1), and inoculum size is 5%; 31 ℃, pH be 7.0 and dissolved oxygen be to control automatically in 5L under 20% condition to cultivate 15h in the fermentor tank to the logarithmic growth middle and later periods, the inoculum size by 20% inserts and contains fermention medium [glucose 12%, ammonium sulfate 0.8%, corn steep liquor 1.5%, soya-bean cake hydrolyzed solution 3.5%, MgSO
47H
2O 0.2%, FeSO
47H
2O 0.003%, MnSO
4H
2O 0.003%, KH
2PO
40.2%, V
B10.00002%, V
H0.00002%.Transfer pH to 7.0～7.2 with NaOH and hydrochloric acid, 115 ℃ the sterilization 15min] 5L control in the fermentor tank automatically, segmentation control leavening temperature: 0～45h is 31 ℃, 46～60h is 28 ℃, feed suitable air, regulate the agitation as appropriate rotating speed, adopting the control of oxygen supply pattern stage by stage dissolved oxygen: 0～20h is 25%, 20～60h is 15%, control pH 7.0～7.2 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 2.0% with residual sugar, fermenting to 60h stops.The output of L-Isoleucine is 31.8g/L, and glucose acid invert ratio is 20.5%.
Claims (4)
1. the method for an efficient fermentative production L-Isoleucine, comprise seed culture and carry out aerobic 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, the temperature of employing are 28-32 ℃.
3. method according to claim 1 is characterized in that: described dissolved oxygen of fermentation liquid concentration is control stage by stage: 0～23h is 10-30%, and 23～60h is 5-20%.
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 8～12%, (NH
4)
2SO 0.4～0.8%, corn steep liquor 1～1.5%, soya-bean cake hydrolyzed solution 3～3.5%, MgSO
47H
2O 0.1～0.2%, FeSO
47H
2O0.002～0.003%, MnSO
40.002～0.003%, KH
2PO
40.1～0.2%, V
B100001～0.00002%, V
H0.00001～0.00002%.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450815A (en) * | 2014-11-20 | 2015-03-25 | 河南巨龙生物工程股份有限公司 | Fermentation method for improving yield of isoleucine |
| CN106701853A (en) * | 2016-12-02 | 2017-05-24 | 武汉远大弘元股份有限公司 | Corynebacterium glutamicum fermentation culture medium and corynebacterium glutamicum fermentation culture method for producing L-isoleucine |
| CN107699595A (en) * | 2017-11-28 | 2018-02-16 | 绍兴厚普生物科技有限责任公司 | A kind of method of Production by Microorganism Fermentation L hydroxyprolines |
| CN108893502A (en) * | 2018-06-28 | 2018-11-27 | 无锡晶海氨基酸股份有限公司 | A method of improving l-Isoleucine yield |
| CN110396493A (en) * | 2019-09-09 | 2019-11-01 | 廊坊梅花生物技术开发有限公司 | The method of culture medium combination and production isoleucine |
| CN110551772A (en) * | 2019-09-21 | 2019-12-10 | 冯世红 | method for improving L-isoleucine yield |
| CN110607330A (en) * | 2019-10-16 | 2019-12-24 | 冯世红 | Production process of L-isoleucine |
| CN111944857A (en) * | 2020-07-22 | 2020-11-17 | 新泰市佳禾生物科技有限公司 | Fermentation method for improving L-isoleucine yield |
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| CN114606275A (en) * | 2022-02-11 | 2022-06-10 | 安徽丰原发酵技术工程研究有限公司 | Method for producing L-isoleucine through fermentation |
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| CN104450815A (en) * | 2014-11-20 | 2015-03-25 | 河南巨龙生物工程股份有限公司 | Fermentation method for improving yield of isoleucine |
| US11319563B2 (en) | 2016-12-02 | 2022-05-03 | Wuhan Grand Hoyo Co., Ltd. | L-isoleucine-producing corynebacterium glutamicum fermentation medium and culture method |
| CN106701853B (en) * | 2016-12-02 | 2019-09-20 | 武汉远大弘元股份有限公司 | A kind of production l-Isoleucine Corynebacterium glutamicum fermentation medium and cultural method |
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| CN110396493B (en) * | 2019-09-09 | 2021-09-07 | 廊坊梅花生物技术开发有限公司 | Culture medium composition and method for producing isoleucine |
| CN110396493A (en) * | 2019-09-09 | 2019-11-01 | 廊坊梅花生物技术开发有限公司 | The method of culture medium combination and production isoleucine |
| CN110551772A (en) * | 2019-09-21 | 2019-12-10 | 冯世红 | method for improving L-isoleucine yield |
| CN110551772B (en) * | 2019-09-21 | 2023-03-28 | 新疆阜丰生物科技有限公司 | Method for improving L-isoleucine yield |
| CN110607330A (en) * | 2019-10-16 | 2019-12-24 | 冯世红 | Production process of L-isoleucine |
| CN110607330B (en) * | 2019-10-16 | 2023-03-10 | 新疆阜丰生物科技有限公司 | Production process of L-isoleucine |
| CN111944857A (en) * | 2020-07-22 | 2020-11-17 | 新泰市佳禾生物科技有限公司 | Fermentation method for improving L-isoleucine yield |
| CN111944857B (en) * | 2020-07-22 | 2021-09-14 | 新泰市佳禾生物科技有限公司 | Fermentation method for improving L-isoleucine yield |
| CN112029683A (en) * | 2020-08-31 | 2020-12-04 | 天津科技大学 | A kind of glucose control technology for improving L-isoleucine yield |
| CN114606275A (en) * | 2022-02-11 | 2022-06-10 | 安徽丰原发酵技术工程研究有限公司 | Method for producing L-isoleucine through fermentation |
| CN114606275B (en) * | 2022-02-11 | 2024-08-13 | 安徽丰原发酵技术工程研究有限公司 | Method for producing L-isoleucine through fermentation |
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