阅读说明：本技术 一种生产和提取色氨酸的工艺 (Process for producing and extracting tryptophan ) 是由 张宗华 边恩来 赵仁楷 王飞 韩富江 于 2019-10-16 设计创作，主要内容包括：本发明属于生物技术领域,公开了一种生产和提取色氨酸的工艺,其包括发酵工序和提取工序。本发明对生产工艺进行了进一步改进,在提高色氨酸产量的同时,保证了流加效果；采用膜技术进行改进色氨酸发酵液传统提取工艺,通过脱色膜去除发酵液中的菌体蛋白,澄清发酵液；通过脱色膜分级去除大分子蛋白和色素,最后采用浓缩膜在常温无相变下进行浓缩。(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 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.)

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 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.

2. The process according to claim 1, wherein the parameters of the fermentation culture are: the rotation speed is 300-: controlling the temperature 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 according to claim 1, wherein the fermentation step comprises feeding a culture medium to increase the tryptophan production, and comprises the following steps:

1) after fermentation culture for about 30h, feeding dipotassium phosphate solution into the fermentation tank at the 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 2.0-4.0 ml/h in each liter of fermentation liquid after fermentation culture for about 30h until the fermentation is finished.

5. The process of claim 4, wherein the dipotassium hydrogen phosphate solution is at a concentration of 5.0% (w/v).

6. The process of claim 4, wherein the ammonium sulfate solution has a concentration of 10.0% (w/v).

7. The process according to claim 4, wherein the concentration of the aqueous arginine solution is 5.0% (w/v).

8. The process according to claim 4, wherein the concentration of the malonic acid and the trifluoroacetic acid in the mixed aqueous solution is 10-20% (v/v) and the concentration of the trifluoroacetic acid is 10-20% (v/v).

9. The process of claim 1, wherein the ceramic membrane is Al₂O₃Microfiltration membrane with cut-off molecular weight of 2000-10000 Da.

10. 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 ℃.

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 C₁₁H₁₂O₂N₂Molecular weight 204.21, nitrogen content 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, is 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 in the plate-frame filtration, the decolorization rate is 80 percent, and the recovery value of filter residue is lowThe mother liquor of the secondary crystallization is turbid and difficult to recoverThe 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-: 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) after fermentation culture for about 30h, feeding dipotassium phosphate solution into the fermentation tank at the 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 2.0-4.0 ml/h in each liter of fermentation liquid after fermentation culture for about 30h until the fermentation is finished.

Preferably, the concentration of the dipotassium phosphate solution is 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 Al₂O₃Microfiltration membrane, with a molecular weight cut-off of 5000 Da.

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 foreign proteins and pigments are reduced, the addition amount is small, the growth of microorganisms is maintained by the slow-acting nitrogen source and the fed-batch quick-acting nitrogen source, the fermentation medium is relatively clean, the energy circulation and transfer generated by thallus metabolism are smooth, most energy is supplied for self growth, and acetic acid is generated less;

controlling the glucose uptake rate is of great significance to 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 in the middle and later stages of fermentation at a lower level so as to achieve the purposes of controlling acetic acid growth 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 of the non-oxidized pentose phosphate cycle is increased, the generation amount of byproducts such as acetic acid and the like in the TCA cycle is reduced, and the yield of the 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 dissolved oxygen, the stirring speed or the air flow is adjusted to maintain the dissolved oxygen at a high level, after the return of dissolved oxygen, the stirring speed or the air flow is adjusted at a fixed time interval, and the sugar supplement speed is increased, so that the dissolved oxygen is controlled to be different levels in the early fermentation stage (0-12 h) and the late fermentation stage (13 h-completion of culture), thereby accelerating the growth rate of strains and avoiding the uncontrolled fermentation caused by excessive sugar supplement due to too fast adjustment of 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: influence of the Mixed aqueous solution 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.