CN109486875A - A kind of zymotechnique producing L-Leu - Google Patents
A kind of zymotechnique producing L-Leu Download PDFInfo
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- CN109486875A CN109486875A CN201811558323.7A CN201811558323A CN109486875A CN 109486875 A CN109486875 A CN 109486875A CN 201811558323 A CN201811558323 A CN 201811558323A CN 109486875 A CN109486875 A CN 109486875A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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Abstract
The invention belongs to technical field of amino acid production, disclose a kind of zymotechnique for producing L-Leu, it includes the following steps: to access seed liquor in fermentation medium, 36.8-37.0 DEG C of cultivation temperature, controlling medium pH by 2 ammonium hydroxide of auto-feeding is 7.0-7.2, by stirring and Ventilation Control dissolved oxygen in 25%-30%；When fermentation is to 12h, compositing acid solution stream is added in tank；When fermentation to for 24 hours when, stop stream plus compositing acid solution, nutrition liquid stream is added in tank, before fermentation ends stoppings in 6 hours stream adds nutrient solution；It in fermentation process, is defoamed with GPE, total fermentation time 48h obtains L-Leu fermentation liquid.The present invention improves the yield of L-Leu by optimization fermentation culture conditions and parameter.
Description
Technical field
The invention belongs to technical field of amino acid production, and in particular to a kind of zymotechnique for producing L-Leu.
Background technique
L-Leu ((2R) -2-amino-3,3-dimethylbutanoic acid) chemical name is L-2- ammonia -4- first
Base valeric acid, white crystals or crystalline powder；It is odorless, mildly bitter flavor.It is readily soluble in formic acid, it is slightly molten in water, in ethyl alcohol or ether
Middle soluble,very slightly.Density is 1.038g/cm3, fusing point is greater than 300 DEG C.
L-Leu, l-Isoleucine and Valine are all branched-chain amino acids, they peomote the flesh after training
Meat restores.Wherein L-Leu is a kind of most effective branched-chain amino acid, and muscle loss can be effectively prevented, because it can be more
Fast decomposition and inversion is glucose.
Currently, L-Leu production method is mainly fermentation method, the L-Leu fermentation liquid of fermentation method production by extracting,
Be concentrated by evaporation, centrifugation drying and etc. finished product can just be made.Patented technology before applicant is " a kind of to utilize sequential simulated shifting
The method that dynamic bed chromatography continuously extracts leucine ", discloses the technique of fermentation leucine, including by Corynebacterium glutamicum seed liquor
It is mixed with bacillus subtilis seed liquor according to certain volume ratio, obtains seed mixture liquid, be then transferred in fermentation medium
Culture, 30 DEG C of temperature, pH value 7.2 incubation time 60 hours, obtains leucine fermentation liquid；This method is compared with single fermentation mode phase
Than improving the efficiency that fermentation produces leucine, cumbersome but there are mixed fermentation technologies, fermentation parameter, which is difficult to control accurately, etc. lacks
It falls into；Chinese invention patent " CN104404495A " discloses a kind of method of fermenting and producing L-Leu, and this method passes through optimization
Fermentation medium and fermentation parameter etc., leucine yield can reach 38g/L in fermentation liquid, however, this method is small-sized fermentation
Tank pilot process expands the effect of fermented and cultured there are more uncertainty, and fermentation and acid amount still have it is to be hoisted.
Summary of the invention
To solve the problems, such as that L-Leu fermentation and extraction technique exists, the present invention provides a kind of L-Leus that produces
Zymotechnique.
The present invention is achieved by the following technical solution:
A kind of zymotechnique producing L-Leu comprising following steps: seed liquor is accessed with the inoculum concentration of 8-10% and is fermented
In culture medium, 36.8-37.0 DEG C of cultivation temperature, controlling medium pH by the ammonium hydroxide of auto-feeding 20% is 7.0-7.2, is passed through
Stirring and Ventilation Control dissolved oxygen are in 25%-30%；When fermentation is to 12h, compositing acid solution stream is added in tank, is maintained in tank
The concentration of glutamic acid is 30-50mg/L；When fermentation to for 24 hours when, stop stream plus compositing acid solution, nutrition liquid stream is added into tank
Interior, maintaining sugared concentration in tank is 0.6-0.9g/L, the stream of stopping in 6 hours plus nutrient solution before fermentation ends；In fermentation process, with GPE
Defoaming, total fermentation time 48h obtain L-Leu fermentation liquid.
Further, the component of the fermentation medium are as follows: glucose 50g/L, (NH4)2SO410g/L, corn pulp 10g/
L, citric acid 0.5g/L, KH2PO44g/L, MgSO4·7H2O 1.5g/L, MnSO4·H2O 10mg/L, FeSO4·7H2O
0.1g/L, VB15mg/L, VH 0.1mg/L。
Further, the component of the compositing acid solution includes glutamic acid, isoleucine and valine.
Further, the component of the compositing acid solution are as follows: glutamic acid 10g/L, isoleucine 5/L, valine 5g/
L。
Further, the component of the nutrient solution includes glucose, glycerol and glycine betaine.
Further, the component of the nutrient solution are as follows: glucose 100g/L, glycerol 5g/L, glycine betaine 1g/L.
Compared with prior art, the beneficial effect that the present invention obtains mainly includes but is not limited to the following aspects:
The present invention optimizes fermented and cultured step by analyzing leucine metabolic pathway；Glycine betaine is containing there are three activity
Methyl has facilitation in terms of L-Leu synthesis；Glycerol provides carbon skeleton, promotes the synthesis of leucine；It is glutamic acid, different bright
Propylhomoserin and valine metabolic pathway can shunt leucine approach, can be to corresponding metabolism by adding above-mentioned amino acid
Approach generates feedback inhibition, so that metabolic fluxes more flow to leucine, the study find that, it is above-mentioned in fermentation initial stage addition
Amino acid, yield effect to leucine are simultaneously little, it may be possible to because fermentation initial stage amino acid synthesis pathway is more faint, not
It can be to bringing greater impact, therefore, selection fermentation 12h is added；And carbon source early period is more sufficient, bacterial strain is increased with biomass
It adds as leading, therefore, selection is being added nutrient solution for 24 hours, to promote the synthesis of leucine；Fermentation medium, compounded amino
A variety of fermentation factors such as acid solution and nutrient solution mutually cooperate with, and improve the fermentation yield of leucine, shorten fermentation time.
Detailed description of the invention
Fig. 1: influence of the glycerol concentration to L-Leu yield in nutrient solution；
Fig. 2: influence of the beet alkali concentration to L-Leu yield in nutrient solution.
Specific embodiment
In order to make those skilled in the art better understand the technical solutions in the application, having below in conjunction with the application
Body embodiment more clearly and completely describes the present invention, it is clear that described embodiment is only the application one
Divide embodiment, instead of all the embodiments.Based on the embodiment in the application, those of ordinary skill in the art are not making
Every other embodiment obtained, should fall within the scope of the present invention under the premise of creative work.
Embodiment 1
A kind of zymotechnique producing L-Leu comprising following steps:
The component of fermentation medium: glucose 50g/L, (NH4)2SO410g/L, corn pulp 10g/L, KH2PO44g/L,
MgSO4·7H2O 1.5g/L, citric acid 0.5g/L, MnSO4·H2O 10mg/L, FeSO4·7H2O 0.1g/L, VB15mg/L,
VH 0.1mg/L。
The component of compositing acid solution: glutamic acid 10g/L, isoleucine 5/L, valine 5g/L,
The component of nutrient solution: glucose 100g/L, glycerol 5g/L, glycine betaine 1g/L；
By taking Corynebacterium glutamicum CGMCC No.7033 as an example, with 10% inoculum concentration by seed liquor (OD600Value is 9.5) access hair
In ferment culture medium, 36.8 DEG C of cultivation temperature, controlling medium pH by the ammonium hydroxide of auto-feeding 20% is 7.2, by stirring and leading to
Air control dissolved oxygen is 25%；When fermentation is to 12h, compositing acid solution stream is added in tank, maintains the concentration of glutamic acid in tank
For 40mg/L；When fermentation to for 24 hours when, stop stream plus compositing acid solution, nutrition liquid stream is added in tank, maintain tank in sugar it is dense
Degree is 0.8g/L, and the stream of stopping in 6 hours adds before fermentation ends；In fermentation process, defoamed with GPE, total fermentation time be 48h to get
L-Leu fermentation liquid.
Embodiment 2
A kind of zymotechnique producing L-Leu comprising following steps:
The component of fermentation medium: glucose 50g/L, (NH4)2SO410g/L, corn pulp 10g/L, citric acid 0.5g/L,
KH2PO44g/L, MgSO4·7H2O 1.5g/L, MnSO4·H2O 10mg/L, FeSO4·7H2O 0.1g/L, VB15mg/L, VH
0.1mg/L。
The component of compositing acid solution: glutamic acid 10g/L, isoleucine 5/L, valine 5g/L,
The component of nutrient solution: glucose 100g/L, glycerol 5g/L, glycine betaine 1g/L；
With 8% inoculum concentration by seed liquor (OD6009.5) value is accesses in fermentation medium, and 37.0 DEG C of cultivation temperature, by certainly
Dynamic stream plus 20% ammonium hydroxide control medium pH are 7.1, by stirring and Ventilation Control dissolved oxygen 30%；It, will when fermentation is to 12h
Compositing acid solution stream is added in tank, and maintaining the concentration of glutamic acid in tank is 30mg/L；When fermentation to for 24 hours when, stop stream plus
Compositing acid solution adds nutrition liquid stream in tank, and maintaining sugared concentration in tank is 0.7g/L, stops within 6 hours before fermentation ends
Stream adds；It in fermentation process, is defoamed with GPE, total fermentation time is 48h to get leucine fermentation liquid.
Embodiment 3
Influence of the fermentation factor to L-Leu yield and Fungal biodiversity in fermentation liquid.
OD value measurement: after fermentation liquid dilutes certain multiple, with 752 spectrophotometric determinations at 620 nm of wavelength.
Contents of Amino Acids in fermentation liquid: it is measured using amino-acid analyzer.
1, influence of the amino acid composition to L-Leu yield and Fungal biodiversity, fermentation method in compositing acid solution
Referring to embodiment 1, concrete outcome is shown in Table 1:
Table 1
| Group | Thallus OD620nm | L-Leu yield g/100ml |
| Glutamic acid+isoleucine+valine | 45.7 | 4.38 |
| Glutamic acid+isoleucine | 45.3 | 4.12 |
| Isoleucine+valine | 45.6 | 3.99 |
| Glutamic acid+valine | 45.8 | 4.04 |
Conclusion: each group Fungal biodiversity does not have notable difference, and L-Leu volume variance is larger in fermentation liquid, wherein paddy
Propylhomoserin+isoleucine+valine group is compared with isoleucine+valine group, L-Leu output increased 0.39g/
100ml improves 9.8 percentage points, 6.3 percentage points is improved compared with glutamic acid+isoleucine group, compared with glutamic acid+valine group
Improve 8.4 percentage points.
2, the influence of glycerol and glycine betaine to L-Leu yield and Fungal biodiversity in nutrient solution, fermentation method is referring to real
Example 1 is applied, concrete outcome is shown in Table 2:
Table 2
| Group | Thallus OD620nm | L-Leu yield g/100ml |
| Glycerol+glycine betaine | 45.7 | 4.38 |
| Glycerol | 42.5 | 3.97 |
| Glycine betaine | 45.1 | 4.13 |
| Do not add glycerol and glycine betaine | 41.3 | 3.81 |
Conclusion: by table 2 it is found that glycerol has facilitation to Fungal biodiversity and L-Leu yield, and glycine betaine is to bacterium
Body biomass has not significant impact, but can be improved L-Leu yield；Compared with not adding the group of glycerol and glycine betaine,
Fungal biodiversity of the invention improves 10.6 percentage points, and 14.9 percentage points of L-Leu output increased.
3, the influence of glycerol and glycine betaine additive amount to L-Leu yield in fermentation liquid.
1) influence of the glycerol concentration to L-Leu yield in nutrient solution, setting concentration are 0,1,2,3,4,5,6,7,8(g/
L), as shown in Figure 1, with glycerol concentration increase, L-Leu yield gradually increases, and after increasing to 4g/L, amplification slows down,
After increasing to 5g/L, L-Leu does not increase significantly, it is contemplated that cost selects glycerol concentration the most suitable for 5g/L.
2) influence of the beet alkali concentration to L-Leu yield in nutrient solution, setting concentration are 0,0.2,0.4,0.6,0.8,
1.0,1.2,1.4,1.6(g/L), as shown in Fig. 2, with beet alkali concentration increase, L-Leu yield gradually increases, and works as battalion
After the concentration of glycine betaine increases to 1g/L in nutrient solution, L-Leu content does not increase in fermentation liquid.
Listed above is only best specific embodiment of the invention.It is clear that the invention is not restricted to which above embodiments, may be used also
With there are many deformations.All changes that those skilled in the art directly can export or associate from present disclosure
Shape is considered as protection scope of the present invention.
Claims (6)
1. a kind of zymotechnique for producing L-Leu comprising following steps: with the inoculum concentration of 8-10% by Corynebacterium glutamicum
Seed liquor accesses in fermentation medium, and 36.8-37.0 DEG C of cultivation temperature, controlling medium pH by auto-feeding ammonium hydroxide is 7.0-
7.2, by stirring and Ventilation Control dissolved oxygen in 25%-30%；When fermentation is to 12h, compositing acid solution stream is added in tank,
The concentration for maintaining glutamic acid in tank is 30-50mg/L；When fermentation to for 24 hours when, stop stream plus compositing acid solution, by nutrient solution
Stream is added in tank, and maintaining sugared concentration in tank is 0.6-0.9g/L, the stream of stopping in 6 hours plus nutrient solution before fermentation ends；Fermentation process
In, it is defoamed with GPE, total fermentation time 48h obtains L-Leu fermentation liquid.
2. the method according to claim 1, wherein the component of the fermentation medium are as follows: glucose 50g/L,
(NH4)2SO410g/L, corn pulp 10g/L, KH2PO44g/L, MgSO4·7H2O 1.5g/L, citric acid 0.5g/L, MnSO4·
H2O 10mg/L, FeSO4·7H2O 0.1g/L, VB15mg/L, VH 0.1mg/L。
3. the method according to claim 1, wherein the component of the compositing acid solution include glutamic acid,
Isoleucine and valine.
4. according to the method described in claim 3, it is characterized in that, the component of the compositing acid solution are as follows: glutamic acid
10g/L, isoleucine 5/L, valine 5g/L.
5. the method according to claim 1, wherein the component of the nutrient solution includes glucose, glycerol and sweet tea
Dish alkali.
6. according to the method described in claim 5, it is characterized in that, the component of the nutrient solution are as follows: glucose 100g/L, glycerol
5g/L, glycine betaine 1g/L.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5069290A (en) * | 1973-10-20 | 1975-06-10 | ||
| CN103031265A (en) * | 2013-01-11 | 2013-04-10 | 新泰市佳禾生物科技有限公司 | Corynebacterium glutamicum mutant strain and application thereof in production of L-leucine by fermentation method |
| CN105886431A (en) * | 2016-04-27 | 2016-08-24 | 天津科技大学 | Corynebacterium glutamicum and method for producing high-yield isoleucine with same |
| CN108841758A (en) * | 2018-07-13 | 2018-11-20 | 大连医诺生物股份有限公司 | Corynebacterium glutamicum mutant and its application in L-Leu production |
-
2018
- 2018-12-19 CN CN201811558323.7A patent/CN109486875B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5069290A (en) * | 1973-10-20 | 1975-06-10 | ||
| CN103031265A (en) * | 2013-01-11 | 2013-04-10 | 新泰市佳禾生物科技有限公司 | Corynebacterium glutamicum mutant strain and application thereof in production of L-leucine by fermentation method |
| CN105886431A (en) * | 2016-04-27 | 2016-08-24 | 天津科技大学 | Corynebacterium glutamicum and method for producing high-yield isoleucine with same |
| CN108841758A (en) * | 2018-07-13 | 2018-11-20 | 大连医诺生物股份有限公司 | Corynebacterium glutamicum mutant and its application in L-Leu production |
Non-Patent Citations (3)
| Title |
|---|
| GROEGER 等: "Microbial production of L-leucine from a-ketoisocaproate by Corynebacterium glutamicum", 《APPL MICROBIOL BIOTECHNOL》 * |
| 张伟国 等: "微生物发酵法生产L-亮氨酸的研究进展", 《食品与生物技术学报》 * |
| 陈宁 等: "柠檬酸钠对L-亮氨酸发酵代谢流分布的影响", 《高校化学工程学报》 * |
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