CN105316371A - Tryptophan fermentation yield increase method - Google Patents
Tryptophan fermentation yield increase method Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial fermentation and particularly relates to a tryptophan fermentation yield increase method. The technical scheme includes that the tryptophan fermentation yield increase method includes the steps of preparing fermentation culture broth and adding L-phenylalanine into the fermentation culture broth; subjecting the fermentation culture broth to sterilization; inoculating to ferment and performing fed-batch fermentation culture. The tryptophan fermentation yield increase method is characterized in that the L-phenylalanine is added into the fermentation culture broth, wherein the optimum adding quantity is 0.2-0.6g/L. According to the technical scheme, the tryptophan fermentation yield increase method has the advantages that tryptophan yield can be increased obviously, transformation rate can be increased, generation of byproducts can be reduced, and production capacity can be improved, so that cost is lowered.
Description
Technical field
The present invention relates to technical field of microbial fermentation, particularly relating to a kind of method for improving tryptophane fermentation yield.
Background technology
Tryptophane (Tryptophan), tryptophane is white or micro-yellow crystal or crystalline powder; Odorless, mildly bitter flavor.Slightly soluble in water, in ethanol soluble,very slightly, insoluble in chloroform, easily molten in formic acid, dissolve in sodium hydroxide test solution or dilute hydrochloric acid.Tryptophane is the precursor substance that in plant materials, growth hormone biosynthesizing is important, and its structure is similar to IAA, ubiquity in higher plant.
The production of L-Trp mainly relies on chemical synthesis and proteolysis method to manufacture the earliest.With the development of the research to microbial method production tryptophane, people start to utilize microbial method fermentative production tryptophane, have now moved towards practical and have been in dominant position.Microbial method can be divided into microbe fermentation method and enzymatic synthesis substantially.Have also appeared direct fermentation and chemical synthesis in recent years, direct fermentation and conversion method combine and produce the research of tryptophane.In addition, the technology such as genetically engineered, enzyme immobilizatio and high-density culture has greatly promoted the process of industrialization of direct fermentation and Production by Enzymes tryptophane in Microbial Breeding and the industrial application of enzyme.
But utilize fermentable production tryptophane but to there is the shortcomings such as by product is many, low conversion rate, therefore, provide a kind of method for improving tryptophane fermentation yield necessary.
Summary of the invention
The object of this invention is to provide a kind of improve tryptophane by adding L-Phe in nutrient solution fermentation yield, increase transformation efficiency, reduce by product and generate, improve throughput, thus the method reduced costs, realize in the following way: in fermentation culture, add L-Phe.
Preferably, the invention provides a kind of method for improving tryptophane fermentation yield, specifically comprising the steps:
(1) prepare fermentation culture and add L-Phe in described fermentation culture;
(2) in the fermentation culture adding L-Phe, access bacterial classification and carry out fermentation culture.
Wherein, described bacterial classification is Recombinant organism CICC10303, and described Recombinant organism CICC10303 is purchased from Chinese industrial Culture Collection.
Wherein, the inoculum size of described bacterial classification is 3%-8%, is preferably 5%.
Wherein, described fermentation culture obtains with water preparation by comprising following component: glucose 8-11g/L, potassium primary phosphate 2-4g/L, yeast powder 1-2g/L, magnesium sulfate 3-4g/L, citric acid 3-4g/L, ferrous sulfate 0.1-0.4g/L.
As preferred scheme, described fermentation culture obtains with water preparation by comprising following component: glucose 8-11g/L, potassium primary phosphate 2-4g/L, yeast powder 1-2g/L, magnesium sulfate 3-4g/L, citric acid 3-4g/L, ferrous sulfate 0.1-0.4g/L, copper sulfate 0.001-0.002g/L, S-WAT 0.02-0.05g/L, cobalt chloride 0.002-0.003g/L.
As most preferred scheme, described fermentation culture obtains with water preparation by comprising following component: glucose 8g/L, potassium primary phosphate 2g/L, yeast powder 1g/L, magnesium sulfate 3g/L, citric acid 3g/L, ferrous sulfate 0.1g/L, copper sulfate 0.001g/L, S-WAT 0.02g/L, cobalt chloride 0.002g/L.
Wherein, the fermentation condition of described fermentation culture is: pressure: 0.03-0.08MPa, pH:6.4-6.7, air quantity: 0.8-1.5vvm, dissolved oxygen 15-25%, the cycle: 45-60h.Be preferably pressure 0.05MPa, air quantity 1.1vvm;
Wherein, the add-on of described L-Phe is 0.2-0.6g/L.After contriver finds to add L-Phe, the output of tryptophane and transformation efficiency all more do not add wants high, and along with the increase of L-Phe addition, output and transformation efficiency are in increasing trend gradually, when addition reaches 0.6g/L, output and transformation efficiency reach maximum; When addition reaches 0.9g/L, although output and transformation efficiency more do not add still want high, its numerical value is all lower than the addition of 0.6g/L, and therefore the addition of L-Phe is 0.2-0.6g/L.
As preferred embodiments of the present invention, a kind of method for improving tryptophane fermentation yield, before being also included in fermentation, pretreated step is carried out to the fermentation culture adding L-Phe, described pretreated concrete grammar is: use sodium hydroxide to regulate the pH of fermentation culture to be 6-7, under 115-125 DEG C of condition, sterilizing 20-40min, is then cooled to 30-37 DEG C.
As preferred embodiments of the present invention, in culturing process, in order to ensure carrying out smoothly of fermenting process, the glucose content scope that the mode adding glucose by stream controls in fermentation culture is 0.1-0.2g/L, in a kind of concrete embodiment, the glucose that stream adds is the D/W of massfraction 60%-70%.
As the most preferred scheme of the present invention, a kind of method for improving tryptophane fermentation yield, comprises the steps:
(1) fermentation culture is prepared and sterilizing: described fermentation culture to be prepared with water obtain by being comprised following component: glucose 8g/L, potassium primary phosphate 2g/L, yeast powder 1g/L, magnesium sulfate 3g/L, citric acid 3g/L, ferrous sulfate 0.1g/L, copper sulfate 0.001g/L, S-WAT 0.02g/L, cobalt chloride 0.002g/L, L-Phe 0.2-0.6g/L, then pH is regulated to be 6-7, under 115-125 DEG C of condition, sterilizing 20-40min, is then cooled to 30-37 DEG C;
(2) ferment: according to the inoculum size of 5%, Recombinant organism is inoculated in described fermentation culture, according to pressure: 0.03-0.08MPa; PH:6.4-6.7; Air quantity: 0.8-1.5vvm; The CMC model 45-60h of dissolved oxygen 15-25%, in culturing process, further stream adds the D/W that massfraction is 60%-70%, to maintain the glucose content scope of fermentation culture for 0.1-0.2g/L.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
For improving a method for tryptophane fermentation yield, comprise the steps:
(1) fermentation culture sterilizing is prepared: glucose 8g/L, potassium primary phosphate 2g/L, yeast powder 1g/L, magnesium sulfate 3g/L, citric acid 3g/L, ferrous sulfate 0.1g/L, copper sulfate 0.001g/L, S-WAT 0.02g/L, cobalt chloride 0.002g/L, L-Phe 0.2g/L is soluble in water; Use sodium hydroxide to regulate the pH to 6.5 of above-mentioned fermentation culture, then sterilizing 30min under 121 DEG C of conditions, after be cooled to 34 DEG C;
(2) inoculation culture: Recombinant organism CICC10303 inoculation enters in fermentation culture by the inoculum size according to 5%, regulated the pH value of fermentation culture by sodium hydroxide in fermenting process, make it maintain 6.4-6.7, the D/W being added 60% by stream makes glucose content maintain 0.1-0.2g/L, in addition, hierarchy of control pressure 0.05MPa, air quantity 1.1vvm, DO15-25%, through the cultivation of 54h, tryptophane reaches 36.8g/L, and transformation efficiency reaches 14%.
Embodiment 2
For improving a method for tryptophane fermentation yield, comprise the steps:
(1) fermentation culture sterilizing is prepared: glucose 8g/L, potassium primary phosphate 2g/L, yeast powder 1g/L, magnesium sulfate 3g/L, citric acid 3g/L, ferrous sulfate 0.1g/L, copper sulfate 0.001g/L, S-WAT 0.02g/L, cobalt chloride 0.002g/L, L-Phe 0.4g/L is soluble in water; Use sodium hydroxide to regulate the pH to 6.5 of above-mentioned fermentation culture, then sterilizing 30min under 121 DEG C of conditions, after be cooled to 34 DEG C;
(2) inoculation culture: Recombinant organism CICC10303 inoculation enters in fermentation culture by the inoculum size according to 5%, regulated the pH value of fermentation culture by sodium hydroxide in fermenting process, make it maintain 6.4-6.7, the D/W being added 65% by stream makes glucose content maintain 0.1-0.2g/L, in addition, hierarchy of control pressure 0.08MPa, air quantity 1.5vvm, DO15-25%, through the cultivation of 50h, tryptophane reaches 40.2g/L, and transformation efficiency reaches 14.6%.
Embodiment 3
For improving a method for tryptophane fermentation yield, comprise the steps:
(1) fermentation culture sterilizing is prepared: glucose 8g/L, potassium primary phosphate 2g/L, yeast powder 1g/L, magnesium sulfate 3g/L, citric acid 3g/L, ferrous sulfate 0.1g/L, copper sulfate 0.001g/L, S-WAT 0.02g/L, cobalt chloride 0.002g/L, L-Phe 0.6g/L is soluble in water; Use sodium hydroxide to regulate the pH to 6.5 of above-mentioned fermentation culture, then sterilizing 30min under 121 DEG C of conditions, after be cooled to 34 DEG C;
(2) inoculation culture: Recombinant organism CICC10303 inoculation enters in fermentation culture by the inoculum size according to 5%, regulated the pH value of fermentation culture by sodium hydroxide in fermenting process, make it maintain 6.4-6.7, the D/W being added 70% by stream makes glucose content maintain 0.1-0.2g/L, in addition, hierarchy of control pressure 0.08MPa, air quantity 1.2vvm, DO15-25%, through the cultivation of 48h, tryptophane reaches 46.8g/L, and transformation efficiency reaches 16.2%.
The existing technique of comparative example 1
A fermentation process for tryptophane, comprises the steps:
(1) fermentation culture sterilizing is prepared: glucose 8g/L, potassium primary phosphate 2g/L, yeast powder 1g/L, magnesium sulfate 3g/L, citric acid 3g/L, ferrous sulfate 0.1g/L, copper sulfate 0.001g/L, S-WAT 0.02g/L, cobalt chloride 0.002g/L is soluble in water; Use sodium hydroxide to regulate the pH to 6.5 of above-mentioned fermentation culture, then sterilizing 30min under 121 DEG C of conditions, after be cooled to 34 DEG C;
(2) inoculation culture: Recombinant organism CICC10303 inoculation enters in fermentation culture by the inoculum size according to 5%, regulated the pH value of fermentation culture by sodium hydroxide in fermenting process, make it maintain 6.4-6.7, the D/W being added 65% by stream makes glucose content maintain 0.1-0.2g/L, in addition, hierarchy of control pressure 0.08MPa, air quantity 1.5vvm, DO15-25%, through the cultivation of 54h, tryptophane is 35.2g/L, and transformation efficiency is 13.6%.
Comparative example 2 investigates the add-on of L-Phe to the impact improving tryptophane output
A fermentation process for tryptophane, comprises the steps:
(1) fermentation culture sterilizing is prepared: glucose 8g/L, potassium primary phosphate 2g/L, yeast powder 1g/L, magnesium sulfate 3g/L, citric acid 3g/L, ferrous sulfate 0.1g/L, copper sulfate 0.001g/L, S-WAT 0.02g/L, cobalt chloride 0.002g/L, L-Phe 0.9g/L is soluble in water; Use sodium hydroxide to regulate the pH to 6.5 of above-mentioned fermentation culture, then sterilizing 30min under 121 DEG C of conditions, after be cooled to 34 DEG C;
(2) inoculation culture: Recombinant organism CICC10303 inoculation enters in fermentation culture by the inoculum size according to 5%, regulated the pH value of fermentation culture by sodium hydroxide in fermenting process, make it maintain 6.4-6.7, the D/W being added 65% by stream makes glucose content maintain 0.1-0.2g/L, in addition, hierarchy of control pressure 0.08MPa, air quantity 1.5vvm, DO15-25%, through the cultivation of 48h, tryptophane is 38.4g/L, and transformation efficiency is 14.3%.
Although above with general explanation, embodiment and test, the present invention is described in detail, and on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. for improving a method for tryptophane fermentation yield, it is characterized in that, in fermentation culture, adding L-Phe.
2. method according to claim 1, is characterized in that, comprises the steps:
(1) prepare fermentation culture and add L-Phe in described fermentation culture;
(2) in the fermentation culture adding L-Phe, access bacterial classification and carry out fermentation culture.
3. method according to claim 1 and 2, is characterized in that: the add-on of described L-Phe is 0.2-0.6g/L.
4. method according to claim 2, is characterized in that: described bacterial classification is Recombinant organism CICC10303.
5. method according to claim 1 and 2, is characterized in that: described fermentation culture obtains with water preparation by comprising following component: glucose 8-11g/L, potassium primary phosphate 2-4g/L, yeast powder 1-2g/L, magnesium sulfate 3-4g/L, citric acid 3-4g/L, ferrous sulfate 0.1-0.4g/L; Be preferably: glucose 8g/L, potassium primary phosphate 2g/L, yeast powder 1g/L, magnesium sulfate 3g/L, citric acid 3g/L, ferrous sulfate 0.1g/L.
6. method according to claim 5, is characterized in that: described fermentation culture also comprises following component: copper sulfate 0.001-0.002g/L, S-WAT 0.02-0.05g/L, cobalt chloride 0.002-0.003g/L, prepares with water; Be preferably: copper sulfate 0.001g/L, S-WAT 0.02g/L, cobalt chloride 0.002g/L.
7. method according to claim 2, it is characterized in that: before being also included in fermentation, pretreated step is carried out to the fermentation culture adding L-Phe, described pre-treatment is specially: regulate the pH of described fermentation culture to be 6-7, under 115-125 DEG C of condition, sterilizing 20-40min, is then cooled to 30-37 DEG C.
8. method according to claim 2, is characterized in that: the condition of described fermentation culture is: pressure: 0.03-0.08MPa, pH:6.4-6.7, air quantity: 0.8-1.5vvm, dissolved oxygen 15-25%, the cycle: 45-60h; Be preferably: pressure 0.05MPa; Air quantity 1.1vvm.
9. method according to claim 1, is characterized in that: in culturing process, and stream adds the D/W that massfraction is 60%-70%, to maintain the content range of glucose for 0.1-0.2g/L.
10. method according to claim 2, is characterized in that: comprise the steps:
(1) fermentation culture is prepared and sterilizing: the component that described fermentation culture comprises is glucose 8g/L, potassium primary phosphate 2g/L, yeast powder 1g/L, magnesium sulfate 3g/L, citric acid 3g/L, ferrous sulfate 0.1g/L, copper sulfate 0.001g/L, S-WAT 0.02g/L, cobalt chloride 0.002g/L, L-Phe 0.2-0.6g/L, said components is prepared with water, then pH is regulated to be 6-7, under 115-125 DEG C of condition, sterilizing 20-40min, is then cooled to 30-37 DEG C;
(2) ferment: Recombinant organism is inoculated CICC10303 in described fermentation culture, according to pressure: 0.03-0.08MPa; PH:6.4-6.7; Air quantity: 0.8-1.5vvm; The CMC model 45-60h of dissolved oxygen 15-25%, in culturing process, further stream adds the D/W that massfraction is 60%-70%, to maintain the glucose content scope of fermentation culture for 0.1-0.2g/L.
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Cited By (3)
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| CN106755159A (en) * | 2016-12-21 | 2017-05-31 | 天津科技大学 | A kind of liquor fermentation culture medium and the method for improving L tryptophan yield |
| CN106967761A (en) * | 2017-05-27 | 2017-07-21 | 新疆苏源生物工程有限公司 | L phenylalanines and preparation method thereof |
| CN107058417A (en) * | 2016-06-27 | 2017-08-18 | 通辽梅花生物科技有限公司 | A kind of tryptophan upgrading synergy new technology |
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| CN106967761A (en) * | 2017-05-27 | 2017-07-21 | 新疆苏源生物工程有限公司 | L phenylalanines and preparation method thereof |
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