CN108893502A - A method of improving l-Isoleucine yield - Google Patents
A method of improving l-Isoleucine yield Download PDFInfo
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- CN108893502A CN108893502A CN201810688752.XA CN201810688752A CN108893502A CN 108893502 A CN108893502 A CN 108893502A CN 201810688752 A CN201810688752 A CN 201810688752A CN 108893502 A CN108893502 A CN 108893502A
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- C—CHEMISTRY; METALLURGY
- 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 discloses a kind of methods for improving l-Isoleucine yield, belong to technical field of microbial fermentation.Traditional nitrogen source (such as corn pulp) in fermentation medium is replaced with the compound nitrogen source being formulated according to a certain percentage by beancake powder, cottonseed protein, brewer's wort, wheat germ powder and casein with fermenting and producing l-Isoleucine by the present invention；Compound nitrogen source of the invention successfully will can be increased to 18g/L from 13g/L with the yield of brevibacterium lactofermentum (Brevibacterium lactofermentation) fermenting and producing l-Isoleucine.
Description
Technical field
The present invention relates to a kind of methods for improving l-Isoleucine yield, belong to technical field of microbial fermentation.
Background technique
L-Isoleucine is one of eight kinds of amino acid needed by human, while being one of three kinds of branched-chain amino acids again, is to close
The raw material of adult body hormone, enzyme has the effect of promoting protein synthesis and it is inhibited to decompose, rises in human life activity
Important function, by extensively transported in biological medicine, organizational project, photochemistry, electrochemistry, food industry, cosmetics etc. lead
Domain.At abroad, l-Isoleucine is also largely used to cow stimulating milk secretion by it, prepares feed addictive and production drinks.
Currently, common Production by Microorganism Fermentation l-Isoleucine.And existing L-isoleucine-producing bacteria mainly has paddy
Propylhomoserin bar bacterium, brevibacterium flavum, Escherichia coli, brevibacterium lactofermentum, with Corynebacterium crenatum and viscous Sai Shi bacillus,
In, in addition to Corynebacterium glutamicum, brevibacterium flavum, remaining equal acid producing ability is very low, only Corynebacterium glutamicum with
The one third of brevibacterium flavum is to half.
For improve brevibacterium lactofermentum, with the acid yield of Corynebacterium crenatum and viscous Sai Shi bacillus etc., had
Angle of many scholars from bacterial strain, it is intended to be sent out by the lactose that mutant strain, building genetic engineering bacterium obtain acid yield raising
Ferment brevibacterium, with Corynebacterium crenatum and viscous Sai Shi bacillus etc..
But since the forward mutation assay rate of mutant strain is extremely low, building of genetic engineering bacterium also has uncertainty, because
This, urgent need finds new raising brevibacterium lactofermentum, with the bacterium acid yield such as Corynebacterium crenatum and viscous Sai Shi bacillus
Thinking.
Summary of the invention
To solve the above problems, the present invention provides a kind of methods for improving l-Isoleucine yield.The method is will to send out
Traditional nitrogen source (such as corn pulp) in ferment culture medium replace with by beancake powder, cottonseed protein, brewer's wort, wheat germ powder and
The compound nitrogen source that casein is formulated according to a certain percentage with fermenting and producing l-Isoleucine, this compound nitrogen source can successfully by with
The yield of brevibacterium lactofermentum (Brevibacterium lactofermentation) fermenting and producing l-Isoleucine from
13g/L is increased to 18g/L.
Technical scheme is as follows：
The present invention provides a kind of method for improving l-Isoleucine yield, the method is by L-isoleucine-producing bacteria
Strain, which is first inoculated in seed culture medium, to spread cultivation, and obtains seed liquor, then seed liquor is inoculated in fermentation medium and is sent out
Ferment obtains fermentation liquid；Nitrogen source in the fermentation medium is compound nitrogen source；The ingredient of the compound nitrogen source include beancake powder,
Cottonseed protein, brewer's wort, wheat germ powder and casein.
In one embodiment of the invention, the beancake powder, cottonseed protein, brewer's wort, wheat germ powder and dry
The mass ratio of casein is (3~4):(0.5~1):(2~3):(2~3):(1~2).
In one embodiment of the invention, the beancake powder, cottonseed protein, brewer's wort, wheat germ powder and dry
The mass ratio of casein is 3:0.5:2:3:1.5.
In one embodiment of the invention, the concentration of the brewer's wort is 20%~35%；Wort concentration refers to
Start the pol of brewer's wort when fermentation.
In one embodiment of the invention, the concentration of the brewer's wort is 25%；Wort concentration, which refers to, to be started to send out
The pol of brewer's wort when ferment.
In one embodiment of the invention, the L-isoleucine-producing bacteria strain is brevibacterium lactofermentum
(Brevibacterium lactofermentation)。
In one embodiment of the invention, the ingredient of the seed culture medium includes 90~100g/L of glucose, sulphur
Sour 12~20g/L of ammonium, 1~2g/L of 90~100g/L of corn pulp, 3~4g/L of potassium dihydrogen phosphate and magnesium sulfate.
In one embodiment of the invention, the ingredient of the seed culture medium includes glucose 96g/L, ammonium sulfate
16g/L, corn pulp 96g/L, potassium dihydrogen phosphate 3.2g/L and magnesium sulfate 1.6g/L.
In one embodiment of the invention, the pH of the seed culture medium is 6~7.
In one embodiment of the invention, the pH of the seed culture medium is 6.8.
In one embodiment of the invention, the condition to spread cultivation is 29~33 DEG C of temperature, 14~20h of time, turns
200~300rpm of speed.
In one embodiment of the invention, the condition to spread cultivation is 31 DEG C of temperature, time 18h, revolving speed 270rpm.
In one embodiment of the invention, the ingredient of the fermentation medium include 2350~3000g/L of glucose,
0~400g/L of ammonium sulfate, 500~750g/L of compound nitrogen source, 0~20g/L of potassium dihydrogen phosphate, 0~10g/L of magnesium sulfate.
In one embodiment of the invention, the ingredient of the fermentation medium includes glucose 2750g/L, ammonium sulfate
280g/L, compound nitrogen source 650g/L, potassium dihydrogen phosphate 15g/L, magnesium sulfate 5g/L.
In one embodiment of the invention, the pH of the fermentation medium is 6~8.
In one embodiment of the invention, the pH of the fermentation medium is 6.7.
In one embodiment of the invention, the condition of the fermentation is 27~36 DEG C of temperature, 60~80h of time, turns
100~150rpm of speed.
In one embodiment of the invention, the condition of the fermentation is 31 DEG C of temperature, time 68h, revolving speed 135rpm.
The present invention provides a kind of above-mentioned l-Isoleucine that the method for improving l-Isoleucine yield produces of application.
The present invention provides a kind of above-mentioned method answering in terms of producing l-Isoleucine for improving l-Isoleucine yield
With.
Beneficial effect：
The present invention replaces with traditional nitrogen source (such as corn pulp) in fermentation medium by beancake powder, cottonseed protein, wheat
The compound nitrogen source that bud juice, wheat germ powder and casein are formulated according to a certain percentage with fermenting and producing l-Isoleucine,
This compound nitrogen source can successfully will be with brevibacterium lactofermentum (Brevibacterium lactofermentation) fermenting and producing
The yield of l-Isoleucine is increased to 18g/L from 13g/L.
Specific embodiment
Below with reference to embodiment and comparative example, the present invention will be further elaborated.
Culture medium involved in following embodiments is as follows：
Seed culture medium：Glucose 96g/L, ammonium sulfate 16g/L, corn pulp 96g/L, potassium dihydrogen phosphate 3.2g/L and sulphur
Sour magnesium 1.6g/L, pH 6.8
Fermentation medium：Glucose 2750g/L, ammonium sulfate 280g/L, compound nitrogen source 650g/L, potassium dihydrogen phosphate 15g/L
And magnesium sulfate 5g/L, pH 6.7
Detection method involved in following embodiments is as follows：
The detection method of l-Isoleucine yield is as follows：
Paper chromatography：
1, point sample：With micro sample applicator, by paper chromatography inspection, 1 μ L of point sample, solvent is n-butanol：Glacial acetic acid：Water=
5:2:1；
2, it is unfolded：Duration of run >=3h, after dry filter paper for chromatography；
3, ninhydrin develops the color：Filter paper for chromatography after drying is infiltrated in the porcelain tray for filling 2% ninhydrin color developing agent, wind
Bluish violet spot is presented in dry 5min in dry 105 DEG C of baking ovens of postposition, and consistent area cuts colour developing spot by size, elution；
4, it elutes：The color spot cut is placed in tool plug test tube with eluant, eluent (75% ethyl alcohol：0.2%Cu5SO45H2O
=39:1), each 5ml is eluted；
5, absorbance measurement：It takes eluent to measure absorbance at 520nm, phase is found on standard curve according to absorbance
The content answered；
6, Specification Curve of Increasing：According to the practical acid production rate of fermentation liquid, the gradient standard sample of corresponding 5 concentration is chosen,
With sample liquid together point sample, expansion, colour developing, elution, absorbance at 520nm is measured, using standard sample as abscissa, absorbance is vertical
Coordinate draws standard curve.
Embodiment 1
Specific step is as follows：
(1) according to the recipe configuration compound nitrogen source of table 1；
(2) brevibacterium lactofermentum (Brevibacterium lactofermentation) bacterium powder is connect with 0.1g/L's
Kind amount, which is inoculated in the 250mL shaking flask equipped with 100mL seed culture fluid, to spread cultivation, and condition is 31 DEG C of temperature, time 18h, turns
Fast 270rpm, obtains seed liquor；
(3) seed liquor is inoculated in the fermented and cultured for containing different composite nitrogen source equipped with 200mL with the inoculum concentration of 0.2g/L
Fermented and cultured is carried out in the 500mL shaking flask of base, condition is 31 DEG C of temperature, time 68h, revolving speed 135rpm, obtains fermentation liquid.
Fermentation liquid is subjected to the detection of l-Isoleucine yield, testing result such as table 2.
1 compound nitrogen source formula of table
| Beancake powder (g/L) | Cottonseed protein (g/L) | Brewer's wort (g/L) | Wheat germ powder (g/L) | Casein (g/L) | |
| A | 195 | 35 | 130 | 195 | 95 |
| B | 195 | 65 | 130 | 195 | 65 |
| C | 195 | 35 | 195 | 130 | 95 |
| D | 230 | 35 | 140 | 150 | 95 |
| E | 190 | 70 | 130 | 190 | 70 |
| F | 180 | 80 | 160 | 130 | 100 |
| G | 180 | 90 | 140 | 130 | 110 |
| H | 150 | 85 | 120 | 120 | 100 |
| I | 200 | 0 | 150 | 150 | 80 |
| J | 200 | 40 | 180 | 180 | 100 |
Wherein, brewer's wort is the brewer's wort of concentration 20%.
Table 2L- isoleucine yield testing result
| L-Isoleucine yield (g/L) | |
| A | 18.4 |
| B | 17.6 |
| C | 18.2 |
| D | 17.2 |
| E | 16.4 |
| F | 15.2 |
| G | 15.1 |
| H | 14.5 |
| I | 14.7 |
| J | 16.2 |
Comparative example 1
By brevibacterium lactofermentum (Brevibacterium lactofermentation) bacterium powder with the inoculation of 0.1g/L
Amount is inoculated in equipped with spreading cultivation in the 250mL shaking flask in 100mL seed culture fluid, and condition is 31 DEG C of temperature, time 18h, turns
Fast 270rpm, obtains seed liquor；Seed liquor is inoculated in using the inoculum concentration of 0.2g/L and is contained equipped with 200mL using corn pulp as nitrogen source
Fermentation medium 500mL shaking flask in carry out fermented and cultured, condition is 31 DEG C of temperature, time 68h, revolving speed 135rpm, is obtained
Fermentation liquid.
Fermentation liquid is subjected to the detection of l-Isoleucine yield, l-Isoleucine yield is 13.2g/L.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of method for improving l-Isoleucine yield, which is characterized in that the method is by L-isoleucine-producing bacteria strain
It is first inoculated in seed culture medium and spreads cultivation, obtain seed liquor, then seed liquor is inoculated in fermentation medium and is fermented,
Obtain fermentation liquid；Nitrogen source in the fermentation medium is compound nitrogen source；The ingredient of the compound nitrogen source includes beancake powder, cottonseed
Albumen, brewer's wort, wheat germ powder and casein.
2. a kind of method for improving l-Isoleucine yield as described in claim 1, which is characterized in that the beancake powder, cotton
Seed albumen, brewer's wort, wheat germ powder and casein mass ratio be (3~4):(0.5~1):(2~3):(2~3):(1
~2).
3. a kind of method for improving l-Isoleucine yield as claimed in claim 1 or 2, which is characterized in that the brewer's wort
Concentration be 20%~35%；Wort concentration refer to start fermentation when brewer's wort pol.
4. a kind of method for improving l-Isoleucine yield a method according to any one of claims 1-3, which is characterized in that the L- is different
Leucine producing strain is brevibacterium lactofermentum (Brevibacterium lactofermentation).
5. a kind of method of raising l-Isoleucine yield as described in claim 1-4 is any, which is characterized in that the seed
The ingredient of culture medium includes 90~100g/L of glucose, 12~20g/L of ammonium sulfate, 90~100g/L of corn pulp, potassium dihydrogen phosphate 3
1~2g/L of~4g/L and magnesium sulfate.
6. a kind of method for improving l-Isoleucine yield a method as claimed in any one of claims 1 to 5, which is characterized in that described to spread cultivation
Condition be 29~33 DEG C of temperature, 14~20h of time, 200~300rpm of revolving speed.
7. a kind of method of raising l-Isoleucine yield as described in claim 1-6 is any, which is characterized in that the fermentation
The ingredient of culture medium includes 2350~3000g/L of glucose, 0~400g/L of ammonium sulfate, 500~750g/L of compound nitrogen source, phosphoric acid
0~20g/L of potassium dihydrogen, 0~10g/L of magnesium sulfate.
8. a kind of method for improving l-Isoleucine yield as claimed in claim 1, which is characterized in that the fermentation
Condition be 27~36 DEG C of temperature, 60~80h of time, 100~150rpm of revolving speed.
9. the different bright ammonia of L- that a kind of method of any raising l-Isoleucine yield of application claim 1-8 produces
Acid.
10. a kind of method of any raising l-Isoleucine yield of claim 1-8 is in terms of producing l-Isoleucine
Application.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1834228A (en) * | 2006-03-30 | 2006-09-20 | 天津科技大学 | Yellow graminic mutant strain and its application in prodn. of L-isoleucine by fementation process |
| US20100120088A1 (en) * | 2001-12-03 | 2010-05-13 | Masato Ikeda | Mutant 6-phosphogluconate dehydrogenase |
| CN101792722A (en) * | 2009-02-04 | 2010-08-04 | 杭州华东医药集团生物工程研究所有限公司 | Streptomyces coelicolor and application thereof |
| CN101962663A (en) * | 2010-11-02 | 2011-02-02 | 天津科技大学 | High-efficiency fermenting method for producing L-isoleucine |
| CN103409476A (en) * | 2013-08-30 | 2013-11-27 | 北京明新高科技发展有限公司 | Fermentation, separation and purification method of L-isoleucine |
-
2018
- 2018-06-28 CN CN201810688752.XA patent/CN108893502A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100120088A1 (en) * | 2001-12-03 | 2010-05-13 | Masato Ikeda | Mutant 6-phosphogluconate dehydrogenase |
| CN1834228A (en) * | 2006-03-30 | 2006-09-20 | 天津科技大学 | Yellow graminic mutant strain and its application in prodn. of L-isoleucine by fementation process |
| CN101792722A (en) * | 2009-02-04 | 2010-08-04 | 杭州华东医药集团生物工程研究所有限公司 | Streptomyces coelicolor and application thereof |
| CN101962663A (en) * | 2010-11-02 | 2011-02-02 | 天津科技大学 | High-efficiency fermenting method for producing L-isoleucine |
| CN103409476A (en) * | 2013-08-30 | 2013-11-27 | 北京明新高科技发展有限公司 | Fermentation, separation and purification method of L-isoleucine |
Non-Patent Citations (5)
| Title |
|---|
| G.E.COLDN等: "Production of isoleucine by overexpression of ilvA in a Corynebacteriumlactofermentum threonine producer", 《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》 * |
| 彭志坚等: "发酵法生产L一异亮氨酸的溶氧控制策略", 《工业微生物》 * |
| 曹凤云: "《食品应用化学》", 31 July 2013, 中国农业大学出版社 * |
| 汪多仁: "《有机食品营养强化剂》", 31 August 2008, 科学技术文献出版社 * |
| 熊宗贵: "《发酵工艺原理》", 31 July 1995, 中国医药科技出版社 * |
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