CN104004798A - Method for producing PHE by microbial fermentation - Google Patents
Method for producing PHE by microbial fermentation Download PDFInfo
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- CN104004798A CN104004798A CN201410277338.1A CN201410277338A CN104004798A CN 104004798 A CN104004798 A CN 104004798A CN 201410277338 A CN201410277338 A CN 201410277338A CN 104004798 A CN104004798 A CN 104004798A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 50
- 230000004151 fermentation Effects 0.000 title claims abstract description 50
- 238000004519 manufacturing process Methods 0.000 title abstract description 10
- 230000000813 microbial effect Effects 0.000 title abstract 2
- 239000000843 powder Substances 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 33
- 238000005457 optimization Methods 0.000 claims abstract description 29
- 125000001477 organic nitrogen group Chemical group 0.000 claims abstract description 24
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 19
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 15
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 15
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 67
- 230000012010 growth Effects 0.000 claims description 42
- 241000588724 Escherichia coli Species 0.000 claims description 23
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- 240000008042 Zea mays Species 0.000 claims description 16
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 16
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 16
- 235000005822 corn Nutrition 0.000 claims description 16
- 230000001580 bacterial effect Effects 0.000 claims description 15
- 239000001888 Peptone Substances 0.000 claims description 13
- 108010080698 Peptones Proteins 0.000 claims description 13
- 235000019319 peptone Nutrition 0.000 claims description 13
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 7
- 238000011218 seed culture Methods 0.000 claims description 6
- 244000068988 Glycine max Species 0.000 claims description 4
- 235000010469 Glycine max Nutrition 0.000 claims description 4
- 235000012343 cottonseed oil Nutrition 0.000 claims description 4
- 235000012054 meals Nutrition 0.000 claims description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 abstract description 62
- 229960004441 tyrosine Drugs 0.000 abstract description 12
- 239000000463 material Substances 0.000 abstract 1
- 230000001502 supplementing effect Effects 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 13
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 7
- 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 description 6
- 239000008103 glucose Substances 0.000 description 6
- 230000000630 rising effect Effects 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 230000001133 acceleration Effects 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
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- QDGAVODICPCDMU-UHFFFAOYSA-N 2-amino-3-[3-[bis(2-chloroethyl)amino]phenyl]propanoic acid Chemical compound OC(=O)C(N)CC1=CC=CC(N(CCCl)CCCl)=C1 QDGAVODICPCDMU-UHFFFAOYSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to an efficient and safe method for producing L-PHE by microbial fermentation, and belongs to the technical field of bioengineering. The concentration of L-PHE in a fermentation liquor is obviously improved through optimization of the concentration of ammonium sulfate and the variety of organic nitrogen sources and a multi-stage L-tyrosine index material supplementing method. The yield of the L-PHE reaches about 56g/L. Meanwhile, the dosage of ammonium sulfate, the dosage of yeast powder and the dosage of the L-tyrosine are obviously reduced compared with a common fermentation method, and the production cost of the L-PHE is greatly lowered.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to the novel method that L-Phe is prepared in a kind of fermentation, the specifically process to fermentative production L-Phe, has carried out the optimization of nitrogenous source and the proposition of multistage index TYR feeding strategy.
Background technology
L-Phe, claim again L-phenylalanine, be needed by human but one of eight kinds of indispensable amino acids that cannot synthesize are voluntarily widely used in Medicines, makeup, food and additive agent field thereof, especially, as one of raw material of synthetic sweetener aspartame.The conventional production methods of L-Phe is chemical synthesis and enzyme process, is raceme mostly but chemical synthesis has environmental pollution, product, subsequent machining technology complexity; Enzyme process has the shortcomings such as raw material costliness.Consider environment and production cost problem, most economical equally also by people widely used be microbe fermentation method.
The production bacterial strain of L-Phe, great majority are that TYR is auxotrophic.In fermenting process, excessive interpolation TYR, can cause the raised growth of cell, thereby makes carbon stream still less flow to the synthetic of L-Phe; The TYR adding is very few, can cause that Growth of Cells is slowly even very few, thereby not have enough thalline to produce more L-Phe.At present, TYR feed supplement has the constant speed of carrying out stream to add, and with good grounds TYR wear rate stream adds, the correlation formula stream of the OUR of with good grounds fermenting process adds, and also has the feed supplement of TYR index.Chinese patent CN102212569A discloses a kind of method that TYR stream that improves L-Phe production intensity adds.After in initial medium, TYR is exhausted, it adds to fermentation ends with 25mg/h-100mg/h constant speed stream.Thus, the present invention proposes multistage index stream and add the strategy of TYR, thereby reach raising L-Phe, reduce the biological production cost of preparing L-Phe, the object of increasing economic efficiency.
In addition, the nitrogenous source of fermenting process, not only maintains the normal growth of cell, and plays an important role in the process of producing L-Phe.Thus, microorganism fermentation is produced in L-Phe process, carries out the optimization of nitrogenous source, comprises the optimization of ammonium sulfate concentrations and organic nitrogen source kind.
Summary of the invention
The object of this invention is to provide a kind of method of fermentative production L-Phe, improve to reach output, and the minimizing of raw material.For achieving the above object, the present invention adopts following technical scheme:
Produce a fermentation process for L-Phe, comprise actication of culture, seed culture, fermentation culture, it is characterized in that, also comprise multistage index TYR feeding strategy.
Further, described method also comprises the optimization of nitrogenous source in fermention medium.
Further, the bacterial strain of described fermentation is e. coli bl21 (pET/P-aspC).
Further, described multistage index TYR feeding strategy concrete steps are as follows: after fermentation 12h, start to add TYR; 12-32h maintains thalline specific growth rate at 0.08h
-1; 32-52h, maintaining thalline specific growth rate is 0.05h
-1; 52-72h, maintaining thalline specific growth rate is 0.02h
-1.
Further, initial TYR concentration is 0-2.4g/L, preferably 0.3-1g/L.
Further, described nitrogenous source optimization comprises the optimization to ammonium sulfate concentrations in fermention medium and organic nitrogen source kind.
Further, the concentration of described ammonium sulfate is 0-30g/L, preferably below 5g/L.
Further, described organonitrogen nitrogenous source be optimized for single organic nitrogen source optimization, its kind and concentration thereof are that yeast powder 10g/L, Dried Corn Steep Liquor Powder 11.5g/L, cottonseed meal 10g/L, soya-bean cake soak powder 7.9g/L, peptone 9.2g/L.
Further, described organonitrogen nitrogenous source be optimized for compound organic nitrogen source optimization, its kind and concentration thereof are yeast powder 5g/L and Dried Corn Steep Liquor Powder 5.75g/L, peptone 4.6g/L and Dried Corn Steep Liquor Powder 5.75g/L, yeast powder 5g/L and peptone 4.6g/L, preferably, be yeast powder and Dried Corn Steep Liquor Powder compound organic nitrogen source.
Further, the total nitrogen of described organic nitrogen source is quite, and in compound organic nitrogen source, two single organic nitrogen sources are to add by its nitrogen content 1:1
Beneficial effect of the present invention is:
The present invention is by the optimization design to nitrogenous source, reduce ammonium sulfate consumption and utilize some cheap organic nitrogen sources to substitute some expensive organic nitrogen sources, reduced the stream dosage of TYR by the proposition of multistage index TYR feeding strategy simultaneously, improve L-Phe output thereby reach, reduce the biological production cost of preparing L-Phe, increase economic efficiency, be conducive to the industry preparation of L-Phe biological process.
Brief description of the drawings
Fig. 1 is that trend map Fig. 2 that under the ammonium sulfate condition of different concns, L-Phe is prepared in e. coli bl21 (pET/P-aspC) fermentation is the trend map (note: YE yeast powder that under different types of organic nitrogen source condition, L-Phe is prepared in e. coli bl21 (pET/P-aspC) fermentation, CSP Dried Corn Steep Liquor Powder, CP cottonseed meal, SP soya-bean cake soaks powder, T peptone, CSPYE yeast powder and Dried Corn Steep Liquor Powder, CSPT peptone and Dried Corn Steep Liquor Powder, YET yeast powder and peptone).
Fig. 3 is under different initial tyrosine conditions, the different specific growth rates of e. coli bl21 (pET/P-aspC) corresponding than the trend map of the synthesis rate of synthetic L-Phe
Fig. 4 (a) and (b) be e. coli bl21 (pET/P-aspC) adopt multistage index TYR feeding strategy, (a) be Different L-tyrosine stream rate of acceleration, the specific growth rate of setting and the time trend of real specific growth rate, (b) time trend of dry cell weight, L-Phe and TYR concentration.Note: F
tyrfor tyrosine flow acceleration, μ is specific growth rate, μ
setfor the specific growth rate of setting
Embodiment
The following examples elaborate to the present invention, but the present invention is not limited.
It is e. coli bl21 (pET/P-aspC) that the present embodiment adopts bacterial strain, (Gong Changbin etc., process engineering journal, the 7th the 3rd phase of volume in 2007,254-258).This starting strain is the disclosed bacterial strain of formerly open source literature that applicant has, and by applicant's preservation voluntarily, applicant is at this statement this starting strain of free dispach (FD) from present patent application day.
Embodiment 1 the present embodiment explanation utilizes e. coli bl21 to produce the method for L-Phe, is preserved by this laboratory.(e. coli bl21 is purchased from Beijing Quanshijin Biotechnology Co., Ltd)
Specifically comprise the following steps:
(1) actication of culture: when slant culture, bacterial strain carries out, after plate streaking, cultivating in constant incubator in slant medium, temperature 30-40 DEG C, activation culture time 1-2d, preserves for seed culture medium inoculation and bacterial strain;
(2) seed culture: add seed culture medium 50mL in 500mL triangular flask, 121 DEG C of sterilizing 20min of high pressure steam, cooling rear access slant medium bacterial strain, culture temperature 30-40 DEG C, shaking speed incubation time 1-2d;
(3) fermentation culture: 1L fermentation cylinder for fermentation culture volume 0.5-0.8L, inoculum size 5%-10% (v/v), temperature 30-40 DEG C, stirring velocity 200-500rpm, air flow is 0.5-0.8vvm.In fermenting process, pass through to add ammoniacal liquor and 2mol/L hydrochloric acid control pH in 7.0 left and right, fermentation time 72h.
Described slant medium/seed culture based formulas is peptone 10g/L, yeast powder 5g/L, NaCl5g/L, pH7.0, and solid medium adds agar 15g/L;
Described fermentative medium formula is glucose 25g/L, (NH
4)
2sO
45g/L, KH
2pO
43g/L, MgSO
43g/L, NaCl1g/L, Trisodium Citrate 1.5g/L, CaCl
22H
2o0.015g/L, FeSO
47H
2o0.1125g/L, L-Tyr0.3g/L, yeast powder 10g/L, vitamins B
10.075g/L, TES1.5mL/L; Wherein TES consists of: Al
2(SO
4)
318H
2o2g/L, CoSO
47H
2o0.75g/L, CuSO
45H
2o0.25g/L, H
3bO
30.5g/L, MnSO
47H
2o24g/L, Na
2moO
42H
2o3g/L, NiSO
46H
2o2.5g/L, ZnSO
47H
2o15g/L; After glucose has consumed, add the glucose of 700g/L, and control glucose concn in 5g/L left and right; When after fermentation 12-18h, carry out TYR feed supplement with the speed of 0.06g/h,
Produce L-Phe content in 2.8g/L left and right.
Embodiment 2 the present embodiment explanation e. coli bl21s (pET/P-aspC) are prepared the optimization method of L-Phe, relate generally to the optimization of ammonium sulfate concentrations
It is e. coli bl21 (pET/P-aspC) that the present embodiment adopts bacterial strain, and the ammonium sulfate concentrations (g/L) in fermention medium is respectively 0,5,10,20,30, and other cultivation and fermentation process are identical with embodiment 1.
In the present embodiment, adopt the ammonium sulfate fermentation of different concns to prepare L-Phe, result as shown in Figure 1.Ammonium sulfate contains thalli growth and the required nitrogen of amino acids production, but the excessive supply of ammonium radical ion can constituting competition property suppress problem.There are some researches show, the ammonium concentration that exceedes 3-5g/L tends to cause restraining effect.The ammonium sulfate of initial 10g/L just plays restraining effect to thalli growth and product L-Phe as shown in Figure 1.The optimal concentration of adding ammonium sulfate in the fermention medium that produces L-Phe is 0-5g/L, now produces L-Phe content in 20g/L left and right.
Embodiment 3 the present embodiment explanation e. coli bl21s (pET/P-aspC) are prepared the optimization method of L-Phe, relate generally to the optimization of organic nitrogen source kind
It is e. coli bl21 (pET/P-aspC) that the present embodiment adopts bacterial strain, and the kind of the organic nitrogen source in fermention medium changes, and other cultivation and fermentation process are identical with embodiment 1.
Above-mentioned organic nitrogen source is respectively single organic nitrogen source and compound organic nitrogen source, and the total nitrogen of the organic nitrogen source adding is suitable; In compound organic nitrogen source, two single organic nitrogen sources add by its nitrogen content 1:1; The kind of single organic nitrogen source and concentration thereof are that yeast powder 10g/L, Dried Corn Steep Liquor Powder 11.5g/L, cottonseed meal 10g/L, soya-bean cake soak powder 7.9g/L, peptone 9.2g/L; Compound organic nitrogen source and concentration thereof are yeast powder 5g/L and Dried Corn Steep Liquor Powder 5.75g/L, peptone 4.6g/L and Dried Corn Steep Liquor Powder 5.75g/L, yeast powder 5g/L and peptone 4.6g/L.
In the present embodiment, adopt different types of organic nitrogen source fermentation to prepare L-Phe, result as shown in Figure 2.In organic nitrogen source, contain multiple amino acids, nucleic acid, trace element etc., be conducive to the growth of cell and the fermentation of L-Phe.As shown in Figure 2, corn steep liquor and yeast powder mixed nitrogen are optimum organic nitrogen sources, and thalli growth and product L-Phe are played an important role, and now produce L-Phe and reach 28.1g/L.
Embodiment 4 the present embodiment explanation e. coli bl21s (pET/P-aspC) are prepared the optimization method of L-Phe, relate generally to the optimization of initial tyrosine concentration
It is e. coli bl21 (pET/P-aspC) that the present embodiment adopts bacterial strain, the initial sugar concentration of fermention medium and initial TYR concentration change and do not carry out the feed supplement of glucose and TYR, and other cultivation and fermentation process are identical with embodiment 1; The concentration of initial TYR is respectively 0,0.3,1,1.7,2.4g/L; And initial sugar concentration is 65g/L.
In the present embodiment, adopt different initial TYR concentration fermentations to prepare L-Phe, result is as shown in table 1.Adopt under the initial tyrosine concentration conditions of difference, different specific growth rates corresponding than the trend of the synthesis rate of synthetic L-Phe as shown in Figure 3.As shown in table 1, along with the rising of TYR concentration, L-Phe content rising, but average L-Phe declines gradually than synthesis rate, average specific growth velocity progressively rises, so show that TYR starting point concentration plays an important role to tyrosine deficient strain in 0.3 left and right the best and TYR: TYR adds very few, Growth of Cells is slow, produce L-Phe also few, TYR adds too much, Growth of Cells is too fast, and relatively few carbon flows to the synthetic of L-Phe.Drawn by Fig. 3,0.3,1, under the initial tyrosine concentration of 2.4g/L, the maximum L-Phe that obtains its correspondence compares synthesis rate; When specific growth rate higher than the maximum L-Phe of correspondence than the specific growth rate of synthesis rate, L-Phe declines along with reducing of specific growth rate than synthesis rate.This may be to drop to specific value when specific growth rate, and carbon stream may be gradually flows to the accumulation of L-Phe from Growth of Cells.Drawn by Fig. 3 again, when specific growth rate is lower than 0.08h
-1, slope of a curve represents the yield of L-Phe to cell; This is a constant, and declines along with the rising of TYR concentration.Draw thus, can control L-Phe than synthesis rate by control ratio growth velocity, and specific growth rate can be controlled by the feed supplement of TYR.
L-Phe is prepared in the different initial TYR concentration fermentations of table 1
Embodiment 5 the present embodiment explanation esherichia coli BL21 (pET/P-aspC) prepare the optimization method of L-Phe, relate generally to the optimization of the index feeding strategy of the constant specific growth rate of TYR
It is e. coli bl21 (pET/P-aspC) that the present embodiment adopts bacterial strain, and the feeding strategy of fermention medium TYR changes, and other cultivation and fermentation process are identical with embodiment 1.
In the present embodiment, adopt the fermentation of index TYR feeding strategy to prepare L-Phe, index feed supplement, according to constant specific growth rate, is respectively 0.02,0.05,0.08h
-1, result is as shown in table 2.Index feed supplement is to make cell arrive the fairly simple feeding strategy easily of one of the specific growth rate of setting.Below the formula of index feed supplement:
In formula (1), F is the speed (L/h) that TYR stream adds, μ
setthe specific growth rate (h setting
-1), X
0and V
0represent separately TYR feed supplement initial cell concentration (g/L) and culture volume (L) before, t is the time (h) of carrying out index feed supplement, Y is the yield of cell to TYR, this is to be obtained by batch fermentation, a constant (g dry cell weight/gL-tyrosine), S
0the concentration of TYR.
As table 2 can draw, along with setting the rising of specific growth rate, dry cell weight, L-Phe content and glucose consumption progressively rise, but its gap is dwindled gradually, the visible rising L-Phe output along with specific growth rate can not rise gradually, and the specific growth rate of setting is controlled at 0.08h by the feed supplement of tyrosine index
-1better; But cell is in different states in the process of fed-batch fermentation, the index TYR feeding strategy of constant specific growth rate is not optimal feeding strategy, so propose the index TYR feeding strategy of multistage specific growth rate.
L-Phe is prepared in the TYR feeding strategy fermentation of the different constant specific growth rate of table 2
Embodiment 6 the present embodiment explanation e. coli bl21s (pET/P-aspC) are prepared the optimization method of L-Phe, relate generally to the proposition of multistage TYR index feeding strategy
The present embodiment adopts bacterial strain and cultivation and fermentation process identical with embodiment 5, adopt and do not add sulfuric acid, use corn steep liquor and yeast powder as organic nitrogen source, and multistage specific growth rate index TYR feeding strategy, after being 12h, the specific growth rate of setting is 0.08h
-1, after 32h, the specific growth rate of setting is 0.05h
-1, after 52h, the specific growth rate of setting is 0.02h
-1.
In the present embodiment, adopt multistage index TYR feeding strategy, Fig. 4 (a) is Different L-tyrosine stream rate of acceleration, the specific growth rate of setting and the time trend of real specific growth rate, the time trend of Fig. 4 (b) dry cell weight, L-Phe and TYR concentration.Adopt this TYR feeding strategy, its specific growth rate fluctuates just near the specific growth rate of setting, dry cell weight and L-Phe content are along with fermentation time is passed growth fast, the final about 56g/L of L-Phe content that produces, greatly improve the output of L-Phe, also reduce the amount of TYR simultaneously.
Contrast before L-Phe optimization is prepared in embodiment 7 the present embodiment explanation e. coli bl21 (pET/P-aspC) fermentations and after optimizing
It is e. coli bl21 (pET/P-aspC) that the present embodiment adopts bacterial strain, and its fermentation is prepared to L-Phe, and carries out the contrast before and after condition optimizing.Fermentation condition before optimization is identical with embodiment 1, and the fermentation condition after optimization is identical with embodiment 6.
In the present embodiment, adopt e. coli bl21 (pET/P-aspC), L-Phe is prepared in the fermentation before and after contrast condition optimizing, as table 3.
Before and after table 3 fermentation condition optimization, prepare the contrast of L-Phe
Embodiment 8 the present embodiment explanation Escherichia coli fermentations are prepared L-Phe
It is intestinal bacteria NT1003 and e. coli bl21 (pET/P-aspC) that the present embodiment adopts bacterial strain, and its fermentation is prepared to L-Phe, and fermentation condition is identical with embodiment 6.
In the present embodiment, adopt intestinal bacteria NT1003 and e. coli bl21 (pET/P-aspC), L-Phe is prepared in the fermentation before and after contrast condition optimizing, as table 4.
Before and after table 4 fermentation condition optimization, prepare the contrast of L-Phe
Claims (10)
1. produce a fermentation process for L-Phe, comprise actication of culture, seed culture, fermentation culture, it is characterized in that, also comprise multistage index TYR feeding strategy.
2. method according to claim 1, is characterized in that, also comprises the optimization of nitrogenous source in fermention medium.
3. according to the method described in claim 1-2, it is characterized in that, the bacterial strain of fermentation is e. coli bl21 (pET/P-aspC).
4. method according to claim 1, is characterized in that, described multistage index TYR feeding strategy concrete steps are as follows: after fermentation 12 h, start to add TYR; 12-32 h maintains thalline specific growth rate at 0.08 h
-1; 32-52 h, maintaining thalline specific growth rate is 0.05 h
-1; 52-72 h, maintaining thalline specific growth rate is 0.02 h
-1.
5. method according to claim 4, is characterized in that, initial TYR concentration is 0-2.4 g/L, preferably 0.3-1 g/L.
6. method according to claim 2, is characterized in that, described nitrogenous source optimization comprises the optimization to ammonium sulfate concentrations in fermention medium and organic nitrogen source kind.
7. method according to claim 6, is characterized in that, the concentration of described ammonium sulfate is 0-30 g/L, preferably below 5 g/L.
8. method according to claim 6, it is characterized in that, described organonitrogen nitrogenous source be optimized for single organic nitrogen source optimization, its kind and concentration thereof are that yeast powder 10g/L, Dried Corn Steep Liquor Powder 11.5g/L, cottonseed meal 10g/L, soya-bean cake soak powder 7.9 g/L, peptone 9.2g/L.
9. method according to claim 6, it is characterized in that, described organonitrogen nitrogenous source be optimized for compound organic nitrogen source optimization, its kind and concentration thereof are yeast powder 5g/L and Dried Corn Steep Liquor Powder 5.75g/L, peptone 4.6g/L and Dried Corn Steep Liquor Powder 5.75g/L, yeast powder 5g/L and peptone 4.6g/L, preferred, be yeast powder and Dried Corn Steep Liquor Powder compound organic nitrogen source.
10. method according to claim 9, is characterized in that, the total nitrogen of described organic nitrogen source is quite, and in compound organic nitrogen source, two single organic nitrogen sources are to add by its nitrogen content 1:1.
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| CN103382492A (en) * | 2007-02-20 | 2013-11-06 | 味之素株式会社 | Method for production of l-amino acid or nucleic acid |
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| CN112251477A (en) * | 2020-11-19 | 2021-01-22 | 乐康珍泰（天津）生物技术有限公司 | Method for improving fermentation yield and sugar-acid conversion rate of L-phenylalanine |
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