CN101245357B - Method for improving shikimic acid yield by fermentation method - Google Patents
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Abstract
A method for improving the yield of a fermentation method for producing shikimic acid pertains to the biochemical field. The invention comprises the steps that: 1) Seed culture: a lactose fermentation bacillus brevis is inoculated in a seed culture medium of pH7.0 to pH7.4, the culture is carried out in a 120 to 180rpm shake bed for 12 to 18 hours at 30 DEG C; the seed culture medium (g/L) includes: 25 to 35 parts of glucose, 15 to 25 parts of ammonium sulfate, 1.0 to 2.0 parts of potassium dihydrogen phosphate, 0.1 to 1.0 part of magnesium sulfate, 0.5 to 2.0 parts of urea, 20 to 30 parts of corn pulp and the rest of water, the pH is regulated to pH 7.0 to pH 7.4, and the sterilization is carried out for 30 minutes at 115 DEG C. 2) Fermentation culture: the seed is inoculated in a fermentation culture medium at pH7.0 to pH7.4, the inoculation amount is 5 percent, the culture is carried out in the 120 to 180rpm shake bed for 48 to 60 hours at 30 DEG C; a glyphosate inhibitor is addedtill the fermentation is going on for 16 to 30 hours; the fermentation culture medium (g/L) includes: 100 to 160 parts of glucose, 30 to 50 parts of ammonium sulfate, 0.5 to 2.0 parts of potassium dihydrogen phosphate, 0.1 to 1.0 part of magnesium sulfate, 20 to 30 parts of corn pulp, 30 to 50 parts of calcium carbonate and the rest of water, the pH is regulated to pH7.0 to pH7.4, and the sterilization is carried out for 30 minutes at 115 DEG C. The yield of the shikimic acid can achieve 1.667 to 2.887 times of the yield before the adding of the glyphosate, and the production is stable and reliable.
Description
Technical field
The present invention relates to a kind of novel method that obtains shikimic acid, particularly obtain the novel method of shikimic acid, belong to biological chemical field by the brevibacterium lactofermentum fermentation.
Background technology
Shikimic acid (shikimic acid) is die aromatischen Aminosaeuren and fragrant vitaminic biosynthesis pathway--the important intermediate of shikimic acid pathway.According to the pharmacological research to shikimic acid, shikimic acid comes anticoagulant by influencing arachidonic acid metabolism, thereby can suppress artery and vein thrombus and cerebral thrombosis; Have anti-inflammatory, analgesic activity.Shikimic acid can also be as antiviral and intermediate cancer therapy drug, is two to dislike mycins, and the synthesis material of antitumor drugs such as lactoyl-glutathione lyase inhibitor also is simultaneously synthetic neuraminidase inhibitor phosphoric acid Ao Simi big (oseltamivir phosphate, trade(brand)name
Crucial starting raw material (Yu Huijie, Zhou Weicheng. the big graphical Synthetic Routes of phosphoric acid Ao Simi. Chinese Journal of Pharmaceuticals, 2006,27 (1)), along with bird flu epidemic situation spreads in the whole world, and the people infects the increase of high pathogenic avian influenza case, and supply falls short of demand more for shikimic acid.
Though shikimic acid pathway extensively exists in plant, microorganism and Parasites, the intermediate metabolites of this approach as the mass production of shikimic acid, quinic acid etc. but and be not easy, mainly is to extract shikimic acid from the Illicium fruit at present.But owing to be subjected to the restriction in the plant resources quantity and the place of production, along with in recent years, bird flu popular, because
Production needs shikimic acid as critical materials, and extraction method has shown slightly and can not meet the needs of production.The chemical synthesis process of shikimic acid and analogue thereof mainly contains Diels.Alder reaction, contrary Diels.Alder reaction, hydrocarbon polymer conversion method and quininic acid conversion method.Chemical process is synthesized shikimic acid, but need expend a large amount of chemical feedstockss, and complex technical process produces objectionable impurities, contaminate environment simultaneously.Therefore, by direct fermentation biosynthesizing shikimic acid, become preferred alternative method.But China also do not have at present can the fermentative production shikimic acid bacterial classification.
In former study, the fermented product of shikimic acid obtains by the Bacillus subtilus of genetic engineering modified intestinal bacteria of multistep or genetic mutation mostly, is included in to block shikimic acid pathway after generating shikimic acid or 3-phosphoric acid shikimic acid in the series of genes operation.(WO 00/0180 for document; WO 2002029078; CN200710110469.0) the reporter gene engineering bacterium fermentation is produced shikimic acid and is knocked out aroL and aroK simultaneously, and it is vitaminic synthetic to have blocked die aromatischen Aminosaeuren and fragrance fully, causes auxotrophy, influences thalli growth.(Fu Xiaohua in Fu Xiaohua research, Gao Yifan, Hao Sijie, influence Deng. intestinal bacteria aroL gene knockout and to the shikimic acid synthetic. Fudan Journal (natural science edition), 2007,46 (3): 366-370.) knock out the aroL gene, same owing to shikimic acid pathway partly is obstructed, thalli growth is bad, and fermentation time is long partially, and the ability that E.coli LKTop strain fermentation is produced shikimic acid is 1.57 of an original strain.In addition, too much genetic manipulation can cause host cell genetic background complexity, and metabolism pressure is excessive; Host cell easily produces reverse mutation simultaneously, causes culturing engineering complicated, and the product productive rate descends.Therefore adopt enzyme inhibitors, the blocking-up shikimic acid pathway impels shikimic acid to accumulate in a large number when biomass is suitable, reduces production of by-products simultaneously, is the preferred method of accumulation shikimic acid.There is not the people that blocker is used for biosynthetic process at present as yet and improves shikimic acid output.
Summary of the invention
The purpose of this invention is to provide a kind of method that improves shikimic acid volume of production with zymotechnics.By in substratum, adding suitable inhibitor, block shikimic acid pathway, thereby improve the tired output of shikimic acid, reduce production of by-products.
It is microbial strains that the present invention adopts brevibacterium lactofermentum, and shikimic acid semi-invariant in seed culture, fermentation culture, detection substratum is characterized in that adding glyphosate as the EPSPS inhibitor in fermentation system.
Concrete steps and method are as follows:
(1) seed culture: brevibacterium lactofermentum is inoculated into the seed culture medium of pH7.0-pH7.4, at 30 ℃, cultivates 12-16h in the 120-180rpm shaking table, the brevibacterium lactofermentum nutrient solution that obtains; Wherein the component of seed culture medium and content are: glucose 25-35g, ammonium sulfate 15-25g, potassium primary phosphate 1.0-2.0g, sal epsom 0.1-1.0g, urea 0.5-2.0g, corn steep liquor 20-30g, water is settled to 1L, transfer pH to pH7.0-pH7.4,115 ℃ of sterilization 30min, liquid amount 25mL/250Ml.
(2) fermentation culture:
To be inoculated into through the brevibacterium lactofermentum nutrient solution that seed culture obtains in the fermention medium of pH7.0-pH7.4,5%, 30 ℃ of inoculum size is cultivated 48-60h in the 120-180rpm shaking table; Fermenting, added concentration to 16-30 hour be that glyphosate between the 1-200 μ moL/L is as synthetase inhibitors;
Wherein fermention medium component and content are: glucose 100-160g, ammonium sulfate 30-50g, potassium primary phosphate 0.5-2.0g, sal epsom 0.1-1.0g, corn steep liquor 20-30g, lime carbonate 30-50g, water is settled to 1L, transfer pH to pH7.0-pH7.4,115 ℃ of sterilization 30min, liquid amount is 25mL/250Ml.
Detect
The fermented liquid 5000rmp that step (2) is obtained is centrifugal, crosses 0.45 μ m millipore filtration, and reversed phase high efficiency liquid phase method detects the output of shikimic acid.
The pH value of above-mentioned seed culture medium and fermention medium adopts prior art alkaline solution commonly used to regulate, as sodium hydroxide solution etc.
The present invention utilizes suitable enzyme inhibitors to improve the method for the synthetic shikimic acid output of fermentation method, because glyphosate has suppressed the conversion of 3-phosphoric acid shikimic acid to 3-phosphoric acid-5-enol pyruvic acid shikimic acid, make shikimic acid pathway after generating the 3-phosphoric acid shikimic acid, be blocked, cause the accumulation of shikimic acid, improve the output of shikimic acid, reduced the production of by-products in shikimic acid pathway downstream simultaneously.Because before adding glyphosate, the growth of brevibacterium lactofermentum after the growth fraction of brevibacterium lactofermentum is partially or completely blocked by genetically engineered is normal, reach the required time shortening of identical biomass, so add glyphosate in due course, both guaranteed that shikimic acid pathway was blocked, guaranteed enough biomasss again.Shikimic acid output can reach 1.667 times-2.887 times when not adding glyphosate.In having avoided simultaneously producing, because the production spawn degeneration problem that the too much genetic manipulation of genetic engineering bacterium causes makes working condition stable more and reliable.
Embodiment
Comparative Examples:
(1) seed culture: brevibacterium lactofermentum is inoculated into seed culture medium, the component of seed culture medium and content are: glucose 30g, ammonium sulfate 20g, potassium primary phosphate 1.5g, sal epsom 0.5g, urea 1.5g, corn steep liquor 25g, water is settled to 1L, transfers pH to pH7.0,115 ℃ of sterilization 30min, liquid amount is 25mL/250Ml.At 30 ℃, cultivate 12h in the 120rpm shaking table.
(2) fermentation culture:
To be inoculated in the fermention medium of pH7.2 according to 5% inoculum size through brevibacterium lactofermentum nutrient solution that seed culture obtains, wherein fermention medium component and content (g/L) are: glucose 130, ammonium sulfate 40, potassium primary phosphate 1.5, sal epsom 0.5, corn steep liquor 25, lime carbonate 40, water is settled to 1L, transfers pH to pH7.2,115 ℃ of sterilization 30min.Fermentation culture adopts the 250ml triangular flask, and liquid amount is 25ml, in 30 ℃, cultivates 48h in the 140rpm shaking table.
(3) detect
The centrifugal 20min of fermented liquid 5000rmp with step (2) obtains crosses 0.45 μ m millipore filtration, and reversed-phased high performace liquid chromatographic detects shikimic acid content, and obtaining shikimic acid content is 0.506gL
-1
Embodiment 1:
(1) seed culture: brevibacterium lactofermentum is inoculated into seed culture medium, the component of seed culture medium and content are: glucose 28g, ammonium sulfate 20g, potassium primary phosphate 1.8g, sal epsom 0.4g, urea 1.8g, corn steep liquor 20g, water is settled to 1L, transfers pH to pH7.0,115 ℃ of sterilization 30min, liquid amount is 25mL/250Ml.At 30 ℃, cultivate 16h in the 120rpm shaking table.
(2) fermentation culture:
To be inoculated in the fermention medium of pH7.0 according to 5% inoculum size through brevibacterium lactofermentum nutrient solution that seed culture obtains, fermention medium component and content are: wherein fermention medium component and content (g/L) are: glucose 140, ammonium sulfate 45, potassium primary phosphate 1.5, sal epsom 1.0, corn steep liquor 20, lime carbonate 30, water is settled to 1L, transfers pH to pH7.0,115 ℃ of sterilization 30min.Fermentation culture adopts the 250ml triangular flask, and liquid amount is 25ml, in 30 ℃, cultivates 60h in the 120rpm shaking table.Adding concentration during fermentation to 30 hour is the glyphosate of 10 μ moL/L.
(3) detect
The centrifugal 20min of fermented liquid 5000rmp with step (2) obtains crosses 0.45 μ m millipore filtration, and reversed-phased high performace liquid chromatographic detects shikimic acid content, and obtaining shikimic acid content is 1.043gL
-1, be 2.061 times when not adding glyphosate.
Embodiment 2:
(1) seed culture: brevibacterium lactofermentum is inoculated into seed culture medium, the component of seed culture medium and content are: the component of seed culture medium and content (g/L) are: glucose 30, ammonium sulfate 20, potassium primary phosphate 1.5, sal epsom 0.5, urea 1.5, corn steep liquor 25, water is settled to 1L, transfers pH to pH7.4,115 ℃ of sterilization 30min, liquid amount is 25mL/250Ml.At 30 ℃, cultivate 14h in the 140rpm shaking table.
(2) fermentation culture:
To be inoculated in the fermention medium of pH7.4 according to 5% inoculum size through brevibacterium lactofermentum nutrient solution that seed culture obtains, fermention medium component and content are: wherein fermention medium component and content (g/L) are: glucose 130, ammonium sulfate 40, potassium primary phosphate 1.5, sal epsom 0.5, corn steep liquor 25, lime carbonate 40, water is settled to 1L, transfers pH to pH7.4,115 ℃ of sterilization 30min.Fermentation culture adopts the 250ml triangular flask, and liquid amount is 25ml, in 30 ℃, cultivates 54h in the 140rpm shaking table.Adding concentration during fermentation to 24 hour is the glyphosate of 20 μ moL/L.
(3) detect
The centrifugal 20min of fermented liquid 5000rmp with step (2) obtains crosses 0.45 μ m millipore filtration, and reversed-phased high performace liquid chromatographic detects shikimic acid content, and obtaining shikimic acid content is 1.456gL
-1, be 2.877 times when not adding glyphosate.
Embodiment 3
(1) seed culture: brevibacterium lactofermentum is inoculated into seed culture medium, the component of seed culture medium and content are: the component of seed culture medium and content are: glucose 32g, ammonium sulfate 15g, potassium primary phosphate 2.0g, sal epsom 0.8g, urea 0.7g, corn steep liquor 30g, water is settled to 1L, transfers pH to pH7.4,115 ℃ of sterilization 30min, liquid amount is 25mL/250Ml.At 30 ℃, cultivate 12h in the 160rpm shaking table.
(2) fermentation culture:
To be inoculated in the fermention medium of pH7.4 according to 5% inoculum size through brevibacterium lactofermentum nutrient solution that seed culture obtains, fermention medium component and content are: wherein fermention medium component and content are: glucose 160g, ammonium sulfate 32g, potassium primary phosphate 0.8g, sal epsom 0.3g, corn steep liquor 30g, lime carbonate 50g, water is settled to 1L, transfers pH to pH7.4,115 ℃ of sterilization 30min.Fermentation culture adopts the 250ml triangular flask, and liquid amount is 25ml, in 30 ℃, cultivates 48h in the 160rpm shaking table.Adding concentration during fermentation to 20 hour is the glyphosate of 100 μ moL/L.
(3) detect
The centrifugal 20min of fermented liquid 5000rmp with step (2) obtains crosses 0.45 μ m millipore filtration, and reversed-phased high performace liquid chromatographic detects shikimic acid content, and obtaining shikimic acid content is 1.246gL
-1, be 2.462 times when not adding glyphosate.
Embodiment 4
(1) seed culture: brevibacterium lactofermentum is inoculated into seed culture medium, the component of seed culture medium and content are: the component of seed culture medium and content are: glucose 25g, ammonium sulfate 18g, potassium primary phosphate 1.0g, sal epsom 0.1g, urea 0.5g, corn steep liquor 20g, water is settled to 1L, transfers pH to pH7.2,115 ℃ of sterilization 30min, liquid amount is 25mL/250Ml.At 30 ℃, cultivate 12h in the 180rpm shaking table.
(2) fermentation culture:
To be inoculated in the fermention medium of pH7.2 according to 5% inoculum size through brevibacterium lactofermentum nutrient solution that seed culture obtains, fermention medium component and content are: wherein fermention medium component and content are: glucose 100g, ammonium sulfate 30g, potassium primary phosphate 0.5g, sal epsom 0.1g, corn steep liquor 20g, lime carbonate 30g, water is settled to 1L, transfers pH to pH7.2,115 ℃ of sterilization 30min.Fermentation culture adopts the 250ml triangular flask, and liquid amount is 25ml, in 30 ℃, cultivates 48h in the 180rpm shaking table.Adding concentration during fermentation to 16 hour is the glyphosate of 50 μ moL/L.
(3) detect
The centrifugal 20min of fermented liquid 5000rmp with step (2) obtains crosses 0.45 μ m millipore filtration, and reversed-phased high performace liquid chromatographic detects shikimic acid content, and obtaining shikimic acid content is 0.843gL
-1, be 1.667 times when not adding glyphosate.
Embodiment 5
(1) seed culture: brevibacterium lactofermentum is inoculated into seed culture medium, the component of seed culture medium and content are: the component of seed culture medium and content (g/L) are: glucose 30, ammonium sulfate 25, potassium primary phosphate 1.2, sal epsom 0.6, urea 1.2, corn steep liquor 25, water is settled to 1L, transfers pH to pH7.0,115 ℃ of sterilization 30min, liquid amount is 25mL/250Ml.At 30 ℃, cultivate 18h in the 140rpm shaking table.
(2) fermentation culture:
To be inoculated in the fermention medium of pH7.0 according to 5% inoculum size through brevibacterium lactofermentum nutrient solution that seed culture obtains, fermention medium component and content are: wherein fermention medium component and content are: glucose 120g, ammonium sulfate 35g, potassium primary phosphate 1.2g, sal epsom 0.7g, corn steep liquor 24g, lime carbonate 40g, water is settled to 1L, transfers pH to pH7.0,115 ℃ of sterilization 30min.Fermentation culture adopts the 250ml triangular flask, and liquid amount is 25ml, in 30 ℃, cultivates 48h in the 160rpm shaking table.Adding concentration during fermentation to 24 hour is the glyphosate of 150 μ moL/L.
(3) detect
The centrifugal 20min of fermented liquid 5000rmp with step (2) obtains crosses 0.45 μ m millipore filtration, and reversed-phased high performace liquid chromatographic detects shikimic acid content, and obtaining shikimic acid content is 1.401gL
-1, be 2.769 times when not adding glyphosate.
Embodiment 6
(1) seed culture: brevibacterium lactofermentum is inoculated into seed culture medium, the component of seed culture medium and content are: the component of seed culture medium and content are: glucose 35g, ammonium sulfate 25g, potassium primary phosphate 2.0g, sal epsom 1.0g, urea 2.0g, corn steep liquor 30g, water is settled to 1L, transfers pH to pH7.4,115 ℃ of sterilization 30min, liquid amount is 25mL/250Ml.At 30 ℃, cultivate 14h in the 130rpm shaking table.
(2) fermentation culture:
To be inoculated in the fermention medium of pH7.4 according to 5% inoculum size through brevibacterium lactofermentum nutrient solution that seed culture obtains, fermention medium component and content are: wherein fermention medium component and content are: glucose 160g, ammonium sulfate 50g, potassium primary phosphate 2.0g, sal epsom 1.0g, corn steep liquor 30g, lime carbonate 50g, water is settled to 1L, transfers pH to pH7.4,115 ℃ of sterilization 30min.Fermentation culture adopts the 250ml triangular flask, and liquid amount is 25ml, in 30 ℃, cultivates 48h in the 130rpm shaking table.Adding concentration during fermentation to 26 hour is the glyphosate of 200 μ moL/L.
(3) detect
The centrifugal 20min of fermented liquid 5000rmp with step (2) obtains crosses 0.45 μ m millipore filtration, and reversed-phased high performace liquid chromatographic detects shikimic acid content, and obtaining shikimic acid content is 1.384gL
-1, be 2.735 times when not adding glyphosate.
Claims (1)
1. a method that improves the tired output of fermentative Production shikimic acid is characterized in that, may further comprise the steps:
1) seed culture: brevibacterium lactofermentum is inoculated into the seed culture medium of pH7.0-pH7.4,, cultivates 12-18h in the 120-180rpm shaking table, obtain the brevibacterium lactofermentum nutrient solution at 30 ℃;
Wherein the component of seed culture medium and content are: glucose 25-35, ammonium sulfate 15-25, potassium primary phosphate 1.0-2.0, sal epsom 0.1-1.0, urea 0.5-2.0, corn steep liquor 20-30, all the other are water, above content unit is g/L, transfers pH to pH7.0-pH7.4,115 ℃ of sterilization 30min;
2) fermentation culture:
To be inoculated into through the brevibacterium lactofermentum nutrient solution that seed culture obtains in the fermention medium of pH7.0-pH7.4,5%, 30 ℃ of inoculum size is cultivated 48-60h in the 120-180rpm shaking table; Fermenting, added concentration to 16-30 hour be that glyphosate between the 1-200 μ moL/L is as synthetase inhibitors;
Wherein fermention medium component and content are: glucose 100-160, ammonium sulfate 30-50, potassium primary phosphate 0.5-2.0, sal epsom 0.1-1.0, corn steep liquor 20-30, lime carbonate 30-50, all the other are water, above content unit is g/L, transfers pH to pH7.0-pH7.4,115 ℃ of sterilization 30min.
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