CN110592154B - Process for producing and extracting tryptophan - Google Patents
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Abstract
The invention belongs to the technical field of biology, and discloses a process for producing and extracting tryptophan, which comprises a fermentation process and an extraction process. The invention further improves the production process, improves the yield of tryptophan and ensures the fed-batch effect; the traditional extraction process of the tryptophan fermentation liquor is improved by adopting a membrane technology, mycoprotein in the fermentation liquor is removed by a decolorizing membrane, and the fermentation liquor is clarified; macromolecular proteins and pigments are removed by a decoloring membrane in a grading way, and finally, a concentration membrane is adopted to carry out concentration at normal temperature without phase change.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a process for producing and extracting tryptophan.
Background
L-tryptophan has the molecular formula C 11 H 12 O 2 N 2 The molecular weight is 204.21, and the nitrogen content is 13.72%. L-tryptophan is neutral aromatic amino acid containing indolyl, has silky luster, hexagonal flaky self-color crystal, no odor and sweet taste. The solubility in water is 1.14g/L (25 ℃), the product is soluble in dilute acid or dilute alkali, is stable in alkali liquor, can be decomposed in strong acid, is slightly soluble in ethanol, and is insoluble in chloroform and diethyl ether.
L-tryptophan is one of eight essential amino acids in the life activities of human bodies and animals, plays an important role in the growth, development and metabolism of the human bodies and the animals, is called as a second essential amino acid, is a third feed additive amino acid after methionine and lysine, and is widely applied to the feed industry.
The production method of tryptophan successively goes through three methods of a protein hydrolysis method, a chemical synthesis method and a microbiological method, wherein the microbiological method comprises a direct fermentation method, a microbiological transformation method and an enzymatic method. At present, tryptophan fermentation enterprises mostly adopt a fed-batch fermentation mode. This approach requires a high degree of control of the residual glucose concentration in the fermentation broth and dispersion of the make-up sugars (including glucose and liquid sugars). If the concentration of the residual glucose is controlled improperly, the normal metabolism of the thalli is affected, the metabolic pathway of the thalli is changed, the fermentation level and the yield of tryptophan are seriously affected, and even the production of the tryptophan is caused. Therefore, the nutrients in the medium play a decisive role in the growth of the cells and the production of metabolites.
The traditional extraction process of tryptophan is complex, and the process flow is as follows: filtering the fermentation liquor by a plate-and-frame filter, concentrating in vacuum, carrying out coarse crystallization, centrifuging, carrying out coarse product, decoloring by active carbon, recrystallizing, centrifuging, drying, packaging and crushing. In the course of this procedure, the user can,
diatomite filler is required to be added for plate-frame filtration, the decolorization rate is 80 percent, and the recovery value of filter residue is low>
The mother liquor after secondary crystallization is turbid and difficult to recover>
The vacuum concentration is easy to damage effective components and increase product pigment due to overhigh local temperature, and the energy consumption is high.
Due to the above aspects, the overall yield of the tryptophan extraction process is below 75%, the extraction process cost is high, and the process needs to be improved urgently.
Disclosure of Invention
In order to solve the problems and overcome the defects of the prior art, the invention further improves the production process, and ensures the fed-batch effect while improving the yield of tryptophan.
The traditional extraction process of the tryptophan fermentation liquor is improved by adopting a membrane technology, mycoprotein in the fermentation liquor is removed by a decolorizing membrane, and the fermentation liquor is clarified; macromolecular protein and pigment are removed by a decoloration membrane in a grading way, and finally, a concentration membrane is adopted to carry out concentration at normal temperature without phase change.
The purpose of the invention is realized by the following technical scheme:
a process for producing and extracting tryptophan comprises a fermentation process and an extraction process;
the fermentation process comprises the following steps: inoculating the engineering bacteria seed liquid of Escherichia coli producing L-tryptophan in a fermentation tank filled with a fermentation medium in an inoculation amount of 1% -10% for fermentation culture for 40h to obtain a fermentation liquid;
the extraction process comprises the following steps: sterilizing the fermentation liquor, and filtering by a ceramic membrane to obtain a filtrate; pumping the filtrate into a chromatographic system to separate L-tryptophan and other impurities; then, removing pigments and proteins through a decolorizing membrane by filtration, and collecting decolorized solution; and (3) dehydrating and concentrating the obtained decolorized solution by using a reverse osmosis membrane to obtain an L-tryptophan eluent, pumping the L-tryptophan eluent into a four-effect evaporator, carrying out vacuum concentration to one fourth of the original volume, cooling and crystallizing the obtained concentrated solution, centrifuging and drying to obtain an L-tryptophan product.
Further, the parameters of the fermentation culture are as follows: the rotation speed is 300-500rpm, the temperature is 35-37 ℃, the tank pressure is 0.05-0.08MPa, the pH value is controlled to be 6.5-6.8 by ammonia water, the fed-batch concentration is 400-500g/L glucose solution, the residual sugar concentration is controlled to be 1-1.5g/L, and the dissolved oxygen is as follows: controlling the temperature to be 25-30% in 0-12h and 15-20% in 13-40 h.
Further, the components of the fermentation medium are as follows: 20g/L of glucose, 5g/L of soybean peptone, 9g/L of dipotassium phosphate, 3.5g/L of citric acid, 3.0g/L of ammonium sulfate, 0.5g/L of magnesium sulfate heptahydrate, 20mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate and 0.1mg/L of biotin.
Further, the fermentation step of increasing the tryptophan production by feeding a culture medium specifically includes:
1) During fermentation culture for about 30h, feeding dipotassium phosphate solution into the fermentation tank at a flow rate of 1.0-3.0 ml/h in each liter of fermentation liquor until the fermentation is finished;
2) Adding ammonium sulfate solution into fermentation tank at flow rate of 0.5-1.5ml/h in fermentation broth after fermentation culture for about 30 h;
3) During fermentation culture for about 30h, feeding arginine aqueous solution into the fermentation tank at a flow rate of 1.0-2.0ml/h in each liter of fermentation liquor until fermentation is finished;
4) And feeding a mixed aqueous solution of malonic acid and trifluoroacetic acid into the fermentation tank at a flow rate of 2.0-4.0ml/h in each liter of fermentation liquid after fermentation culture for about 30h until the fermentation is finished.
Preferably, the dipotassium hydrogen phosphate solution has a concentration of 5.0% (w/v).
Preferably, the concentration of the ammonium sulfate solution is 10.0% (w/v).
Preferably, the concentration of the arginine aqueous solution is 5.0% (w/v).
Preferably, in the mixed aqueous solution of the malonic acid and the trifluoroacetic acid, the concentration of the malonic acid is 10-20% (v/v), and the concentration of the trifluoroacetic acid is 10-20% (v/v).
Preferably, the ceramic film is Al 2 O 3 Microfiltration membrane, with a molecular weight cut-off of 5000Da.
Preferably, the reverse osmosis membrane is a polyamide composite membrane, the pressure is 0.55MPa, and the temperature is 55 ℃.
The technical scheme of the invention has the following outstanding advantages and uniqueness:
in the fermentation medium, peptone is adopted to replace yeast extract as a slow-acting nitrogen source, complex and unstable substances such as impure proteins and pigments are reduced, the addition amount is less, the growth of microorganisms is maintained by the slow-acting nitrogen source and a fed-batch quick-acting nitrogen source, the fermentation medium is relatively clean, the energy circulation and transfer generated by the metabolism of thalli are smooth, most energy is supplied for the growth of the culture medium, and acetic acid is less generated;
the control of the glucose uptake rate is of great significance for L-tryptophan fermentation. The acetic acid yield is increased due to overhigh glucose concentration at the initial fermentation stage, but the strain proliferation is not facilitated due to overlow concentration; the low glucose intake rate in the middle and later stages of fermentation is beneficial to inhibiting the generation of byproducts such as acetic acid, and the high glucose intake rate increases TCA circulating metabolic flow and the HMP pathway flow is insufficient. The invention adopts relatively high glucose concentration in the early stage of fermentation and controls the glucose concentration at a lower level in the middle and later stages of fermentation so as to achieve the purposes of controlling the growth of acetic acid and maintaining the proliferation efficiency of the strains.
According to the invention, by adding the dipotassium hydrogen phosphate and the ammonium sulfate in a flowing manner in the later stage of fermentation, a nitrogen source and nutrient substances required by the growth of the strain are effectively supplemented, the growth activity of the strain is maintained, and the acid production performance of fermentation is greatly improved; in the middle and later period of fermentation, the metabolic byproducts are increased, and the addition of a proper amount of arginine can improve the activity of the glucose-6-phosphate dehydrogenase and reduce the accumulation of the metabolic byproducts, thereby further improving the yield of the L-tryptophan.
The malonic acid and the trifluoroacetic acid can inhibit key enzymes of the TCA cycle, so that the TCA cycle is weakened, the flow rate of the non-oxidized pentose phosphate cycle is increased, the generation amount of acetic acid and other byproducts in the TCA cycle is reduced, and the yield of L-tryptophan is further increased; however, the TCA cycle is not susceptible to excessive attenuation, which results in significant inhibition of strain growth and thus reduced L-tryptophan production.
The supplemented sugar can be quickly and uniformly distributed in the fermentation liquor, so that the problems of over osmotic pressure of the fermentation liquor and excessive byproduct acetic acid caused by over-high local concentration of the sugar are effectively solved, and the problems that the substrate is limited due to over-low local concentration of glucose in the fermentation liquor, so that residual sugar in the fermentation liquor is quickly exhausted, and the production capacity of strains cannot be exerted to the maximum extent are solved;
before the return of the dissolved oxygen, the stirring speed or the air flow is adjusted to maintain the dissolved oxygen at a high level, and after the return of the dissolved oxygen, the stirring speed or the air flow is adjusted at regular time intervals to increase the sugar supplement rate, so that the dissolved oxygen is controlled to be at different levels in the early fermentation stage (0-12 h) and the late fermentation stage (13 h-completion), thereby accelerating the growth rate of the strains and avoiding the uncontrolled fermentation due to excessive sugar supplement caused by too fast adjustment of the process parameters;
in the extraction process, the membrane separation process is an efficient and environment-friendly separation process, is a multidisciplinary crossed high-new technology, can present various characteristics on physical, chemical and biological properties, and has more advantages; the traditional extraction process of the tryptophan is improved by adopting a membrane separation technology, the overall yield of the tryptophan is greatly improved, the energy consumption is reduced to a half of that of the traditional process, the operation is automatic, the water recycling is realized, the production cost is reduced, and the working environment is improved; the invention has the advantages of high-efficiency separation process, low energy consumption, less wastewater generation amount, energy conservation and emission reduction; the working temperature is close to the room temperature, and the quality stability is good; continuous operation and strong flexibility; pure physical process, and no waste increase.
Drawings
FIG. 1: the effect of dipotassium phosphate feed rate on L-tryptophan production;
FIG. 2: the effect of ammonium sulfate feed rate on L-tryptophan production;
FIG. 3: influence of the addition amount of arginine on the yield of L-tryptophan and the biomass of thalli;
FIG. 4 is a schematic view of: influence of the aqueous mixture of malonic acid and trifluoroacetic acid on the yield of L-tryptophan and biomass of cells.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be described more clearly and completely below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A process for producing and extracting tryptophan comprising the steps of:
1. a fermentation process:
step 1): the L-tryptophan engineering bacterium escherichia coli (E. coli) TRTH, culturing to a bacterium solution with a certain concentration;
step 2): inoculating the bacterial liquid obtained in the step 1) into a shake flask filled with a primary seed culture medium in an inoculation amount of 0.5%, and culturing at 36 ℃ at 100-200rpm for 10-20h;
step 3): inoculating the first-stage shake flask seeds obtained in the step 2) into a seeding tank of a second-stage seed culture medium in an inoculation amount of 5%, controlling the air volume to be 5-10L/min, the rotation speed to be 200-400rpm, the temperature to be 35-37 ℃, the tank pressure to be 0.05-0.08MPa, controlling the pH value to be 6.5-6.8 through ammonia water, and culturing for 10-20h;
step 4): the second-stage fermentation tank seeds (OD) obtained in the step 3) are added 600 Value 11) was inoculated in a fermentation tank containing a fermentation medium at 7% inoculum size at 400rpm at 36 ℃ under 0.06MPa with pH controlled at 6.6 by ammonia, residual sugar concentration controlled at 1g/L by fed-batch glucose solution at 400g/L, dissolved oxygen: the pre-fermentation period (0-12 h) is controlled to 25% and the post-fermentation period (13 h-cultivation end) is controlled to 20%, the fermentation period being 40h.
The first-stage culture medium comprises the following components: 5g/l of yeast powder, 10g/l of tryptone, 10g/l of sodium chloride and 50mg/l of tetracycline hydrochloride, and the pH value is 6.6.
The secondary seed culture medium comprises the following components: 10g/l of dipotassium phosphate, 10g/l of yeast powder,
20g/l glucose, 2g/l disodium hydrogen phosphate, 4g/l tryptone, 1.0 g/l magnesium chloride, 0.2g/l trisodium citrate, 0.005g/l ferrous sulfate heptahydrate, 0.005g/l manganese sulfate monohydrate, pH controlled at 6.6, and sterilizing at 115 ℃ for 15min.
The fermentation medium comprises the following components: 20g/L of glucose, 5g/L of soybean peptone, 9g/L of dipotassium phosphate, 3.5g/L of citric acid, 3.0g/L of ammonium sulfate, 0.5g/L of magnesium sulfate heptahydrate, 20mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate and 0.1mg/L of biotin; the pH was controlled at 6.6 and sterilized at 115 ℃ for 15min. The flow of adding culture solution in the fermentation process is as follows:
1) After about 30h of culture, 5.0% (w/v) of dipotassium hydrogen phosphate solution is fed into the fermentation tank at a flow rate of 2.0ml/h per liter of fermentation liquor until the end of fermentation.
2) After about 30h of cultivation, 10.0% (w/v) ammonium sulphate solution was fed to the fermenter at a flow rate of 1.5ml/h per litre of fermentation broth until the end of the fermentation.
3) After about 30h of culture, 5.0% (w/v) arginine aqueous solution was fed to the fermenter at a flow rate of 2.0ml/h per liter of fermentation broth until the end of fermentation.
4) After about 30h of culture, the mixed aqueous solution of malonic acid and trifluoroacetic acid is fed into the fermentation tank at a flow rate of 4.0ml/h per liter of fermentation broth until the end of fermentation. In the mixed aqueous solution of malonic acid and trifluoroacetic acid, the concentration of malonic acid is 15% (v/v), and the concentration of trifluoroacetic acid is 15% (v/v).
2. An extraction process:
sterilizing the fermentation broth, and treating with ceramic membrane (ceramic membrane is 99% Al by mass) 2 O 3 Microfiltration membrane with molecular weight cutoff of 5000 Da) filtering to obtain filtrate; pumping the filtrate into a chromatographic system to separate L-tryptophan and other impurities;
the primary decolorizing membrane operates, manual pump and valve stopping is strictly forbidden in the operation process, the pressure difference of an air source and a feeding filter is strictly forbidden to be closed and is not more than 0.5bar, and pigment and protein with large molecular weight in filtrate are removed by the primary decolorizing membrane;
and clicking each membrane group to select activation, selecting each pump to an automatic mode to start, and clicking production to start. The system carries out liquid level detection, and when the liquid level meets the requirement of equipment, feeding and external circulation are started for 60s; the sequence starts. After all modules are started, starting a dialysis water pump to add water: the water adding amount is required to be 20-50% (the water adding amount is controlled according to the concentrated solution content required by a workshop), the water adding amount of each module can be adjusted according to the dialysis flux of each module, but the total water adding multiple does not exceed 50% of the feeding flow, and the acid content of the concentrated solution does not exceed 5 per mill;
the secondary decolorizing membrane operates, the filtrate of the primary decolorizing membrane is put into a circulating tank of experimental equipment for secondary decolorizing, and small molecular proteins and pigments are removed, at the moment, the content of impurities is obviously reduced, and the filtrate is pure and transparent and has no visible solid; filtering again, dialyzing and adding water, wherein the water adding amount of each module can be adjusted according to the flux of each module;
and (3) dehydrating and concentrating the obtained decolorized solution by using a reverse osmosis membrane (polyamide composite membrane, the pressure is 0.55MPa, the temperature is 55 ℃) to obtain L-tryptophan eluent, pumping the L-tryptophan eluent into a four-effect evaporator, carrying out vacuum concentration to obtain one fourth of the original volume, cooling and crystallizing the obtained concentrated solution, centrifuging and drying to obtain an L-tryptophan product. The detection shows that the product purity is more than 99 percent, and the product yield is about 90 percent.
Example 2
A process for producing and extracting tryptophan comprising the steps of:
1. a fermentation process:
step 1): the L-tryptophan engineering bacterium escherichia coli (E. coli) TRTH, culturing to a bacterium solution with a certain concentration;
step 2): inoculating the bacterial liquid obtained in the step 1) into a shake flask filled with a primary seed culture medium in an inoculation amount of 0.1-1%, and culturing at 36 ℃ at 100-200rpm for 10-20h;
step 3): inoculating the primary shake flask seeds obtained in the step 2) into a seed tank of a secondary seed culture medium in an inoculation amount of 1-10%, controlling the air volume to be 5-10L/min, the rotation speed to be 200-400rpm, the temperature to be 35-37 ℃, the tank pressure to be 0.05-0.08MPa, controlling the pH value to be 6.5-6.8 through ammonia water, and culturing for 10-20h;
step 4): the second-stage fermentation tank seeds (OD) obtained in the step 3) are added 600 Value of 12) was inoculated in a fermentor containing a fermentation medium at 6% inoculum size at 300rpm, temperature of 35 ℃, pressure of 0.05MPa, pH controlled at 6.5 by ammonia, fed-batch concentration of 500g/L glucose solution controlled at 1.2g/L residual sugar concentration, dissolved oxygen: the pre-fermentation period (0-12 h) is controlled to 30% and the post-fermentation period (13 h-cultivation end) is controlled to 20%, the fermentation period being 40h.
The first-stage culture medium comprises the following components: 5g/l of yeast powder, 10g/l of tryptone, 10g/l of sodium chloride, 100mg/l of tetracycline hydrochloride and 6.7 of pH value.
The secondary seed culture medium comprises the following components: 10g/l of dipotassium phosphate, 10g/l of yeast powder,
30 g/l glucose, 3g/l disodium hydrogen phosphate, 3g/l tryptone, 0.1 g/l magnesium chloride, 0.10 g/l trisodium citrate, 0.001g/l ferrous sulfate heptahydrate, 0.001g/l manganese sulfate monohydrate, pH controlled at 6.7, and sterilization at 115 ℃ for 15min.
The fermentation medium comprises the following components: 20g/L of glucose, 5g/L of soybean peptone, 9g/L of dipotassium phosphate, 3.5g/L of citric acid, 3.0g/L of ammonium sulfate, 0.5g/L of magnesium sulfate heptahydrate, 20mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate and 0.1mg/L of biotin; the pH was controlled at 6.7 and sterilized at 115 ℃ for 15min. The flow of adding culture solution in the fermentation process comprises the following steps:
1) After about 30h of culture, 5.0% (w/v) of dipotassium hydrogen phosphate solution is fed into the fermentation tank at a flow rate of 1.5ml/h per liter of fermentation liquor until the end of fermentation.
2) After about 30h of cultivation, 10.0% (w/v) ammonium sulphate solution was fed to the fermenter at a flow rate of 1ml/h per litre of fermentation broth until the end of the fermentation.
3) After about 30h of culture, 5.0% (w/v) arginine aqueous solution was fed to the fermenter at a flow rate of 1.5ml/h per liter of fermentation broth until the end of fermentation. The yield of tryptophan at this time was 50g/l.
4) After about 30h of culture, the mixed aqueous solution of malonic acid and trifluoroacetic acid is fed into the fermentation tank at a flow rate of 3.0ml/h per liter of fermentation broth until the end of fermentation. In the mixed aqueous solution of the malonic acid and the trifluoroacetic acid, the concentration of the malonic acid is 15% (v/v), and the concentration of the trifluoroacetic acid is 15% (v/v);
2. an extraction process:
sterilizing the fermentation liquor, and filtering by a ceramic membrane to obtain a filtrate; pumping the filtrate into a chromatographic system to separate L-tryptophan and other impurities;
the primary decolorizing membrane operates, manual pump and valve stopping is strictly forbidden in the operation process, the pressure difference of an air source and a feeding filter is strictly forbidden to be closed and is not more than 0.5bar, and pigment and protein with large molecular weight in filtrate are removed by the primary decolorizing membrane;
and clicking each membrane group to select activation, selecting each pump to an automatic mode to start, and clicking production to start. The system carries out liquid level detection, and when the liquid level meets the requirement of equipment, feeding and external circulation are started for 60s; the sequence starts. After all modules are started, starting a dialysis water pump to add water: the water adding amount is required to be 20-50% (the water adding amount is controlled according to the concentrated solution content required by a workshop), the water adding amount of each module can be adjusted according to the dialysis flux of each module, but the total water adding multiple does not exceed 50% of the feeding flow, and the acid content of the concentrated solution does not exceed 5 per mill;
the secondary decolorizing membrane operates, the filtrate of the primary decolorizing membrane is put into a circulating tank of experimental equipment for secondary decolorizing, and small molecular proteins and pigments are removed, at the moment, the content of impurities is obviously reduced, and the filtrate is pure and transparent and has no visible solid; filtering again, dialyzing and adding water, wherein the water adding amount of each module can be adjusted according to the flux of each module;
and (3) dehydrating and concentrating the obtained decolorized solution by using a reverse osmosis membrane to obtain an L-tryptophan eluent, pumping the L-tryptophan eluent into a four-effect evaporator, carrying out vacuum concentration to one fourth of the original volume, cooling and crystallizing the obtained concentrated solution, centrifuging and drying to obtain an L-tryptophan product.
Example 3
1. The influence of the addition amount of dipotassium phosphate on the yield of L-tryptophan in the fermentation liquor.
The fermentation process was the same as in example 1, without the step of using a culture solution. Firstly, the effect of feeding dipotassium hydrogen phosphate solution on the fermentation is verified through experiments. As shown in figure 1, the L-tryptophan content in the fermentation broth is correspondingly increased with the increase of the dipotassium phosphate addition, the flow rate reaches a peak value basically when the flow rate is 2ml/h, the flow rate is continuously increased, the tryptophan yield is not obviously improved, the influence trend of the dipotassium phosphate on the biomass of the bacteria is similar to that of the tryptophan (not shown in the figure), and the main reason is that the biomass of the bacteria can be improved by adding the dipotassium phosphate solution in a flowing manner, so that the tryptophan yield is improved.
2. Influence of the addition of ammonium sulfate on the yield of L-tryptophan in the fermentation broth.
The flow rate of the dipotassium phosphate is selected to be 2ml/h, the addition amount of the ammonium sulfate with different gradients is set, as shown in figure 2, when the flow rate of the ammonium sulfate is 1.5ml/h, the content of the L-tryptophan is 43.9g/L, the flow rate of the ammonium sulfate is continuously increased, the influence on the tryptophan is not great, and the flow rate of 1.5ml/h meets the requirement of the bacterial strain for normal acid production.
3. The influence of the addition amount of arginine on the yield of L-tryptophan in the fermentation broth and the biomass of thalli.
When the flow rate of the dipotassium phosphate is 2ml/h and the flow rate of the ammonium sulfate is 1.5ml/h, the influence of arginine on the yield of L-tryptophan and the biomass of the bacteria is verified. As shown in FIG. 3, the L-tryptophan yield and the biomass of the cells were increased continuously with the increase of the feeding rate of arginine by setting different gradient feeding amounts, and both the L-tryptophan yield and the biomass of the cells reached peak values at a flow rate of 2ml/h, which were increased by 5.01% and 4.75%, respectively, compared with the case where no arginine was added.
The reason is that the metabolic byproducts are increased in the middle and later stages of fermentation, and the addition of a proper amount of arginine can improve the activity of the glucose-6-phosphate dehydrogenase, reduce the accumulation of the metabolic byproducts, reduce the damage to the strain and further improve the yield of the L-tryptophan.
4. The influence of the mixed aqueous solution of malonic acid and trifluoroacetic acid on the yield of L-tryptophan in the fermentation broth and the biomass of the cells.
The flow rate of dipotassium phosphate is 2ml/h, the flow rate of ammonium sulfate is 1.5ml/h, and when the flow rate of arginine is 2ml/h, the mixed aqueous solution of malonic acid and trifluoroacetic acid with different feeding rates is set. The reason may be that the malonic acid and the trifluoroacetic acid can inhibit key enzymes of the TCA cycle, so that the TCA cycle is weakened, the flow of the non-oxidative pentose phosphate cycle is increased, the generation amount of byproducts such as acetic acid and the like in the TCA cycle is reduced, the yield of the L-tryptophan is further increased, and the yield is improved by 6.72%; however, the TCA cycle is not easily weakened excessively, and the strain growth is obviously inhibited due to excessive weakening, so that the yield of the L-tryptophan is reduced. The reason why the yield of tryptophan is improved to a certain extent only by single malonic acid or trifluoroacetic acid, the maximum is 3.3% and 4.1%, respectively, and the effect is far less than that of the two which are used in a synergistic manner is probably that the malonic acid and the trifluoroacetic acid adopt different mechanisms to inhibit the tricarboxylic acid cycle and can intervene in the synthesis of tryptophan more effectively.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. A process for producing and extracting tryptophan comprises a fermentation process and an extraction process;
the fermentation process comprises the following steps: inoculating the engineering bacteria seed liquid of Escherichia coli for producing L-tryptophan in a fermentation tank filled with a fermentation medium in an inoculation amount of 1-10% for fermentation culture for 40h to obtain a fermentation liquid;
the extraction process comprises the following steps: sterilizing the fermentation liquor, and filtering by a ceramic membrane to obtain a filtrate; pumping the filtrate into a chromatographic system to separate L-tryptophan and other impurities; then, removing pigments and proteins through a decolorizing membrane by filtration, and collecting decolorized solution; dehydrating and concentrating the obtained decolorized solution by a reverse osmosis membrane to obtain L-tryptophan eluent, pumping the L-tryptophan eluent into a four-effect evaporator, concentrating the L-tryptophan eluent in vacuum to be one fourth of the original volume, cooling and crystallizing the obtained concentrated solution, centrifuging and drying the concentrated solution to obtain an L-tryptophan product;
in the fermentation process, the yield of tryptophan is improved by feeding culture solution, and the method specifically comprises the following steps:
1) After fermentation culture for 30h, feeding a dipotassium phosphate solution into the fermentation tank at a flow rate of 1.0-3.0 ml/h in each liter of fermentation liquor until the fermentation is finished;
the concentration of the dipotassium phosphate solution is 5.0 percent w/v;
2) After fermentation culture for 30h, feeding ammonium sulfate solution into the fermentation tank at a flow rate of 0.5-1.5ml/h in each liter of fermentation liquor until fermentation is finished;
the concentration of the ammonium sulfate solution is 10.0% w/v;
3) Fermenting and culturing for 30h, and feeding arginine aqueous solution into the fermentation tank at a flow rate of 1.0-2.0ml/h in each liter of fermentation liquor until fermentation is finished;
the concentration of the arginine aqueous solution is 5.0% w/v;
4) Feeding a mixed aqueous solution of malonic acid and trifluoroacetic acid into a fermentation tank at a flow rate of 2.0-4.0ml/h in each liter of fermentation liquor after fermentation culture for 30h until the fermentation is finished;
the mixed aqueous solution of malonic acid and trifluoroacetic acid has a concentration of 10-20% v/v, and a concentration of trifluoroacetic acid of 10-20% v/v.
2. The process according to claim 1, wherein the parameters of the fermentation culture are: the rotation speed is 300-500rpm, the temperature is 35-37 ℃, the tank pressure is 0.05-0.08MPa, the pH value is controlled to be 6.5-6.8 through ammonia water, the concentration of residual sugar is controlled to be 1-1.5g/L through feeding glucose solution with the concentration of 400-500g/L, and the dissolved oxygen is as follows: the temperature is controlled to be 25-30% in 0-12h and 15-20% in 13-40 h.
3. The process of claim 1, wherein the fermentation medium comprises: 20g/L of glucose, 5g/L of soybean peptone, 9g/L of dipotassium phosphate, 3.5g/L of citric acid, 3.0g/L of ammonium sulfate, 0.5g/L of magnesium sulfate heptahydrate, 20mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate and 0.1mg/L of biotin.
4. The process of claim 1, wherein the ceramic membrane is Al 2 O 3 Micro-filtration membrane with molecular weight cutoff of 2000-10000Da.
5. The process of claim 1, wherein the reverse osmosis membrane is a polyamide composite membrane at a pressure of 0.5 to 0.6MPa and a temperature of 50 to 60 ℃.
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