CN111004822A - Production process of high-purity threonine - Google Patents
Production process of high-purity threonine Download PDFInfo
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- 239000004473 Threonine Substances 0.000 title claims abstract description 90
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 28
- 238000000855 fermentation Methods 0.000 claims abstract description 133
- 230000004151 fermentation Effects 0.000 claims abstract description 133
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims abstract description 104
- 229960002898 threonine Drugs 0.000 claims abstract description 89
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- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims description 38
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 5
- 239000006260 foam Substances 0.000 claims description 5
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 5
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 5
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 5
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- 102000001390 Fructose-Bisphosphate Aldolase Human genes 0.000 description 2
- 108010068561 Fructose-Bisphosphate Aldolase Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
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- 239000012138 yeast extract Substances 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
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- 108020003285 Isocitrate lyase Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-L aspartate group Chemical group N[C@@H](CC(=O)[O-])C(=O)[O-] CKLJMWTZIZZHCS-REOHCLBHSA-L 0.000 description 1
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- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 239000004223 monosodium glutamate Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 1
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- 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
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/38—Separation; Purification; Stabilisation; Use of additives
- C07C227/40—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/38—Separation; Purification; Stabilisation; Use of additives
- C07C227/40—Separation; Purification
- C07C227/42—Crystallisation
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Abstract
The invention belongs to the technical field of amino acid fermentation, and discloses a production process of high-purity threonine, which comprises a fermentation process and an extraction and separation process, wherein the fermentation process comprises the following steps: inoculating the L-threonine producing strain seed liquid into a fermentation tank containing a fermentation culture medium for fermentation for 36 hours, and collecting the fermentation liquid; the extraction and separation process comprises the following steps: centrifuging by a high-speed disc separator, filtering by a ceramic membrane, performing primary decolorization treatment by activated carbon, performing secondary decolorization treatment by a decolorization membrane, performing ultrafiltration membrane treatment, and finally concentrating, crystallizing and drying to obtain the product. The production process of the invention has high yield and purity of threonine, and is suitable for industrial popularization and use.
Description
Technical Field
The invention belongs to the technical field of amino acid fermentation, and particularly relates to a production process of high-purity threonine.
Background
The molecular formula of L-threonine is C4H9NO3Relative molecular weight 119.12.L-threonine is white crystal or crystalline powder, is a polar amino acid, and has slightly sweet taste. Melting and decomposing at 253 ℃. Soluble in water at high temperature, with a solubility of 20.5g/100ml at 25 deg.C, insoluble in ethanol, diethyl ether and chloroform. The specific rotation is 27.6-29 degrees, and the isoelectric point PI is 5.64. The threonine industry in China is developed later, basically no production is produced in China before the 90 s of the last century, and the production is started after the 90 s. In recent years, the demand for threonine in domestic and foreign markets has been strongly increasing year by year, the global threonine market has been rapidly increasing in recent years at a rate of more than 20% per year, and the demand in regions such as asia and north america has also been rapidly increasing. The production is pulled vigorously by the demand, in 1993, the yield of threonine worldwide is less than 4000 tons, in 1996 5000 tons, in 1999 to 2.5 ten thousand tons, in 2002 to 4 ten thousand tons, and in 2005 the yield is close to 7 ten thousand tons, which is increased by more than 15 times before 10 years.
Through the development of ten years, the yield cannot break through 200 tons at the end of the 20 th century, threonine belongs to one of products of industrial fermentation, and according to relevant statistics, the worldwide yield of threonine is only 4000 tons in 1993, the yield is increased to 2.5 thousands of tons in 1999, more than 4 thousands of tons in 2002, and more than 20 thousands of tons at present. The rapid development of threonine represents its importance in the field of amino acid fermentation. The increase of 20 percent is increased gradually every year, and the addition amount of threonine is further increased for improving the product quality of the feed industry. The main market of the product is also the pharmaceutical industry, and the product is mainly used for various amino acid transfusions. At present, the main threonine production enterprises in the world are Japan monosodium glutamate company, Germany Degussa company, American ADM company, Japan Co-and fermentation industry company and the like, the yield of the large companies accounts for about 90% of the global share, the capacity reaches about 10 ten thousand tons, the domestic threonine production is in the development stage, as early as 2014, domestic manufacturers have 8 to 9 families, most of the threonine supply domestic requirements, and some companies export amino acids abroad, the domestic production level still has a certain gap with the foreign country in terms of technical indexes, cost indexes and product quality, and the price of the product is slightly higher than the foreign price. In recent two years, with the increase of manufacturers, the production of threonine tends to steadily increase day by day, and the gap between the threonine and foreign manufacturers is gradually reduced.
The requirement for threonine is only dependent on protein raw materials, which contain not only threonine but also other essential and non-essential amino acids, and if threonine is not added to the feed, the amino acid balance of the feed cannot be improved as much as possible, and the formulation cost of the feed cannot be further reduced, so to speak, threonine is a threshold that must be taken to improve the amino acid balance and is a bottleneck property problem that cannot be avoided by all formulations, and the metabolic pathway of threonine in the body is different from other amino acids, and is only catalyzed by dehydrogenase action and transaminage action, but is converted into amino acids of other substances by Threonine Dehydratase (TDH) and Threonine Dehydrogenase (TDG) and aldolase (TDG) and the enzymatic pathway is changed into amino acids of other substances by glycine, and alanine, and glycine through the aldolase, and acetic acid through the enzyme α.
In the production of threonine, most of corn steep liquor and molasses are used as fermentation nitrogen sources, threonine fermentation thalli are directly subjected to threonine extraction after membrane filtration, fermentation by-product mycoprotein is directly sold as cheap mycoprotein after being filtered by a flocculation precipitation plate frame, and the added value of the product is low. The mycoprotein is directly used for preparing the feed, and in combination with the current industrial situation, the Angel yeast utilizes beer yeast, bread yeast and the like to produce high-value-added products such as yeast extract, yeast powder, yeast extract, peptone and the like, so that the yeast powder has higher selling price and larger profit. The prior patent technology of the applicant has carried out more researches on threonine fermentation, including optimization of culture medium and fermentation process; in the prior art, "optimization research on fermentation conditions of L-threonine industrial production, luweining, journal of biology 2010" optimizes fermentation conditions of industrial production, and a series of optimization research on fermentation conditions is performed by taking high-yield L-threonine bacteria e.coli THR6 as an initial strain and combining actual industrial production conditions of the company, and the results show that: adding 0.2% of industrial-grade growth promoter, using complex sugar to replace glucose as initial sugar, controlling the concentration of the initial sugar at 60g/L, and controlling the Dissolved Oxygen (DO) between 10% and 20% in the fermentation process except the growth peak period; the fermentation time is 40h, the final fermentation tank-discharging wet thallus is about 45g/L, and the L-threonine content can reach about 100 g/L.
Disclosure of Invention
On the basis of the prior patent technology 'L-threonine fermentation process optimization method', the invention continuously separates and extracts threonine fermentation liquor, and provides a production process of high-purity threonine.
The invention is realized by the following technical scheme:
the production process of high-purity threonine comprises a fermentation process and an extraction and separation process.
Further, the fermentation process comprises the following steps:
inoculating the L-threonine production strain seed liquid into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 1-2% for fermentation, controlling the stirring speed at 300-37 ℃ and the dissolved oxygen at 500rpm, controlling the dissolved oxygen at 15-20% by ventilation and stirring, defoaming by using a foam killer, and stopping fermentation when the fermentation time is 36h, and collecting the fermentation liquid.
Further, the extraction and separation process comprises the following steps:
centrifuging the fermentation liquid for 3-5min at the centrifugal speed of 4000-; filtering the threonine feed liquid by using a ceramic membrane, and collecting filtrate; performing primary decolorization treatment on the filtrate with activated carbon by plate-type closed filter for 30-90 min; performing secondary decolorization treatment on the decolorized clear liquid in a decolorizing membrane to obtain decolorized clear liquid; passing the decolorized clear liquid through an ultrafiltration membrane, and collecting ultrafiltrate; concentrating the ultrafiltrate to one fourth of the original volume, slowly cooling to 16 deg.C, and adjusting to obtain isoelectric solution; precipitating with isoelectric solution for 6-18 hr, centrifuging, collecting crystallized threonine, and oven drying.
Further, the method comprises the steps of:
the sugar content is controlled to be 2-3% by adding 50% sucrose solution (50 g sucrose dissolved in water to 100ml is 50% solution) until the fermentation is finished.
Further, the method comprises the steps of: the pH value is controlled to be 7.0 by adding 20-30% ammonia water in a flowing manner until the fermentation is finished.
Further, the method comprises the steps of: fermenting for about 20h, and adding hydrogen peroxide into fermentation tank at a flow rate of 2-3ml/h in each liter of fermentation liquid until fermentation is finished.
Further, the method comprises the steps of: fermenting for about 20h, and feeding mixed aqueous solution containing succinic acid and sodium citrate into the fermentation tank at a flow rate of 10-20ml/h per liter of fermentation liquid until the fermentation is finished.
Further, the method comprises the steps of: after fermenting for about 30h, adding chitosan into the fermentation tank at one time, and controlling the concentration of the chitosan to be 40-60 mg/L.
Further, the fermentation medium comprises the following components:
60g/L of sucrose, 30g/L of glucose, 20g/L of corn steep liquor, 5g/L of ammonium sulfate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, VB12mg/L，VH50μg/L。
Preferably, the concentration of the succinic acid and the concentration of the sodium citrate in the mixed aqueous solution are both 50 g/L.
The beneficial effects of the invention mainly comprise the following aspects:
because the threonine begins to accumulate in large quantity after the bacterial strain is fermented for about 10 hours and is fermented for about 20 hours, the feedback inhibition is obvious along with the increase of threonine and byproducts; it is appropriate to select this time to optimize the metabolic synthesis pathway.
During the threonine synthesis process, the threonine is not directly subjected to tricarboxylic acid cycle, oxaloacetate enters an aspartate group for metabolism, threonine is finally generated, certain inhibition effect on citrate dehydrogenase can be generated by feeding sodium citrate, the tricarboxylic acid cycle is properly weakened, the generation of byproducts and the energy loss can be reduced, and thus the method has positive significance for improving the threonine yield;
glyoxylate circularly consumes a large amount of ATP and wastes carbon sources, and the isocitrate lyase is inhibited by adding succinic acid in a flowing manner, so that the flux entering the glyoxylate circulating metabolism is reduced, and the threonine yield is increased;
the invention adopts the combination of hydrogen peroxide feeding and oxygen supply by aeration in the middle and later stages of fermentation, improves the mass transfer rate of oxygen, and can improve the cell density and metabolic efficiency of a fermentation system by certain addition concentration and addition mode.
In the later fermentation period, a certain amount of chitosan is added, the amino group on the chitosan is combined with teichoic acid or lipopolysaccharide with negative charge in the cell wall of the strain and chelates metal cations, so that the permeability of the cell wall is changed, the endocrine of threonine is promoted to the extracellular, and the yield of threonine in the fermentation liquid is improved.
Drawings
FIG. 1: the influence of the addition of hydrogen peroxide on the yield of L-threonine in the fermentation broth;
FIG. 2: the effect of succinic acid and sodium citrate on threonine production;
FIG. 3: the effect of succinic acid and sodium citrate on the conversion of sugar acids;
FIG. 4: influence of Chitosan on the production of L-threonine in fermentation broths.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1
The production process of high-purity threonine comprises the following steps of fermentation and extraction and separation:
a fermentation process:
step 1) preparation of a fermentation medium: 60g/L of sucrose, 30g/L of glucose, 20g/L of corn steep liquor, 5g/L of ammonium sulfate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, VB12mg/L，VH50μg/L。
Step 2) fermentation: inoculating seed solution of L-threonine producing strain (such as Escherichia coli engineering bacteria TRFC) into fermentation tank containing fermentation medium at inoculation amount of 1.5%, fermenting, and inoculating with inoculation density OD600At the temperature of 36 ℃, the stirring speed of 300-500rpm, controlling the dissolved oxygen amount to be 20% by aeration and stirring, defoaming by using a foam killer, stopping fermentation for 36 hours, and collecting fermentation liquor;
in the fermentation process, a feed liquid needs to be fed in a flowing mode, and the method specifically comprises the following steps:
1) controlling the sugar content to be 3% by adding 50% of sucrose solution (50 g of sucrose is dissolved in water until 100ml is a 50% solution) until the fermentation is finished;
2) controlling the pH value to be 7.0 by adding 20% ammonia water until the fermentation is finished;
3) after fermentation is carried out for about 20 hours, adding hydrogen peroxide into the fermentation tank at a flow rate of 2ml/h in each liter of fermentation liquor until the fermentation is finished;
4) after fermentation is carried out for about 20 hours, adding mixed aqueous solution of succinic acid and sodium citrate into the fermentation tank in a feeding flow manner at a flow rate of 15ml/h in each liter of fermentation liquor until the fermentation is finished; in the mixed aqueous solution, the concentrations of succinic acid and sodium citrate are both 50 g/L;
5) after fermenting for about 30 hours, adding chitosan into the fermentation tank at one time, and controlling the concentration of the chitosan to be 40 mg/L;
an extraction and separation process:
centrifuging the fermentation liquor for 3min at a centrifugal speed of 4000rpm by using a high-speed disc separator, and collecting threonine feed liquid and mycoprotein precipitate; filtering threonine feed liquid by using a ceramic membrane (with the molecular weight cutoff of 10000 Da), setting the filtration parameter pressure difference of 0.06MPa and the temperature of 45 ℃, and collecting filtrate; performing primary decolorization treatment on the filtrate with activated carbon by plate-type closed filter for 60 min; performing secondary decolorization treatment on the decolorized clear liquid in a decolorizing membrane, setting decolorizing process parameters of membrane inlet pressure of 0.9MPa, membrane outlet pressure of 0.8MPa and feed liquid flow rate of 100mL/min to obtain decolorized clear liquid; passing the decolorized clear solution through an ultrafiltration membrane (molecular weight cutoff is 300 Da), and collecting ultrafiltrate; concentrating the ultrafiltrate to one fourth of the original volume, slowly cooling to 16 deg.C, and adjusting to pH 6.16 to obtain isoelectric solution; and (3) precipitating the isoelectric solution for 12 hours, centrifuging, collecting crystallized threonine, and drying the crystallized threonine to obtain the threonine. The threonine product has the purity of 96.2 percent and the yield of 84.3 percent by HPLC detection.
Example 2
The production process of high-purity threonine comprises the following steps of fermentation and extraction and separation:
a fermentation process:
step 1) preparation of a fermentation medium: 60g/L of sucrose, 30g/L of glucose, 20g/L of corn steep liquor, 5g/L of ammonium sulfate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, VB12mg/L，VH50μg/L。
Step 2) fermentation: inoculating seed solution of L-threonine producing strain (such as Escherichia coli engineering bacteria TRFC) into fermentation tank containing fermentation medium at inoculation amount of 2%, fermenting, and inoculating with inoculation density OD600At the temperature of 36 ℃, the stirring speed of 300-500rpm, controlling the dissolved oxygen amount to be 15% by aeration and stirring, defoaming by using a foam killer, stopping fermentation for 36 hours, and collecting fermentation liquor;
in the fermentation process, a feed liquid needs to be fed in a flowing mode, and the method specifically comprises the following steps:
1) controlling the sugar content to be 2.5% by feeding 50% of sucrose solution until the fermentation is finished;
2) controlling the pH value to be 7.0 by adding 25% ammonia water until the fermentation is finished;
3) after fermentation is carried out for about 20 hours, adding hydrogen peroxide into the fermentation tank at a flow rate of 3ml/h in each liter of fermentation liquor until the fermentation is finished;
4) after fermentation is carried out for about 20 hours, adding mixed aqueous solution of succinic acid and sodium citrate into the fermentation tank in a feeding flow manner at a flow rate of 10ml/h in each liter of fermentation liquor until the fermentation is finished; in the mixed aqueous solution, the concentrations of succinic acid and sodium citrate are both 50 g/L;
5) after fermenting for about 30 hours, adding chitosan into the fermentation tank at one time, and controlling the concentration of the chitosan to be 60 mg/L;
an extraction and separation process:
centrifuging the fermentation liquor for 3min at a centrifugal speed of 5000rpm by using a high-speed disc separator, and collecting threonine feed liquid and mycoprotein precipitation; filtering threonine feed liquid by using a ceramic membrane (with the molecular weight cutoff of 20000 Da), setting the differential pressure of filtering parameters to be 0.06MPa and the temperature to be 45 ℃, and collecting filtrate; performing primary decolorization treatment on the filtrate with activated carbon by a plate-type closed filter for 90 min; performing secondary decolorization treatment on the decolorized clear liquid in a decolorizing membrane, setting decolorizing process parameters of membrane inlet pressure of 0.9MPa, membrane outlet pressure of 0.8MPa and feed liquid flow rate of 100mL/min to obtain decolorized clear liquid; passing the decolorized clear solution through an ultrafiltration membrane (molecular weight cutoff is 200 Da), and collecting ultrafiltrate; concentrating the ultrafiltrate to one fourth of the original volume, slowly cooling to 16 deg.C, and adjusting to pH 6.16 to obtain isoelectric solution; and (3) precipitating the isoelectric solution for 18 hours, centrifuging, collecting crystallized threonine, and drying the crystallized threonine to obtain the threonine. The threonine product has the purity of 97.9 percent and the yield of 83.1 percent by HPLC detection.
Comparative example
A fermentation process comprising the steps of:
step 1) preparation of a fermentation medium: 60g/L of sucrose, 30g/L of glucose, 20g/L of corn steep liquor, 5g/L of ammonium sulfate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, VB12mg/L，VH50μg/L。
Step 2) fermentation: inoculating seed solution of L-threonine producing strain (such as Escherichia coli engineering bacteria TRFC) into fermentation tank containing fermentation medium at inoculation amount of 1.5%, fermenting, and inoculating with inoculation density OD600At the temperature of 36 ℃, the stirring speed of 300-500rpm, controlling the dissolved oxygen amount to be 20% by aeration and stirring, defoaming by using a foam killer, stopping fermentation for 36 hours, and collecting fermentation liquor;
in the fermentation process, a feed liquid needs to be fed in a flowing mode, and the method specifically comprises the following steps:
1) controlling the sugar content to be 3% by adding 50% of sucrose solution (50 g of sucrose is dissolved in water until 100ml is a 50% solution) until the fermentation is finished;
2) the pH was controlled to 7.0 by feeding 20% ammonia until the end of the fermentation.
Example 3
Influence of various factors on threonine yield and sugar acid conversion rate in fermentation liquor.
1. On the basis of the comparative example, the influence of hydrogen peroxide on fermentation is verified, and the adding flow rate of hydrogen peroxide in each liter of fermentation liquid is set as follows: 0,1, 2, 3, 4,5, unit ml/h, as shown in figure 1, then increasing the flow rate of hydrogen peroxide improves the threonine yield to a certain extent, when the flow rate is lower than 2ml/h, the threonine yield is improved more quickly, the sugar acid conversion rate is not greatly influenced (not shown in the figure), when the flow rate is 2ml/h, the threonine yield approaches the peak value, the flow rate of hydrogen peroxide is continuously increased, and the influence on threonine is not large. The reason is probably that the hydrogen peroxide has positive effect on the proliferation of the thalli, so that the yield of threonine is improved, and the conversion rate of the sugar acid is not greatly influenced.
2. The accelerating rate of hydrogen peroxide flow is selected to be 2ml/h, and the influence of succinic acid and sodium citrate on the fermentation efficiency is verified. Three groups of parallel experiments are set, namely a succinic acid group, a sodium citrate group and a succinic acid and sodium citrate group, the feeding rate is set to be 1, 5, 10,15,20,25 and 30, and the unit is ml/h, as shown in figure 2, the threonine yield is greatly improved by the succinic acid and sodium citrate group and is obviously higher than that of the succinic acid and sodium citrate groups, the feeding rate of 10-20ml/h is selected to greatly improve the threonine yield, the feeding amount is continuously increased, and the influence on the threonine yield is small; as shown in FIG. 3, when the flow acceleration rate is 15ml/h, the sugar-acid conversion rate of the succinic acid and sodium citrate group is the highest, and can reach 46.8 percent, which is 45.1 percent higher than that of the succinic acid group and 45.9 percent higher than that of the sodium citrate group. Succinic acid and sodium citrate promote metabolic flux to flow to a threonine pathway by weakening the pathway which is not beneficial to L-threonine synthesis, so that the sugar acid conversion rate is improved, and the yield of threonine in fermentation liquor is further improved.
3. The influence of chitosan on the yield of threonine is verified by selecting the hydrogen peroxide flow acceleration rate of 2ml/h and the feeding rate of the mixed aqueous solution of succinic acid and sodium citrate of 15 ml/L. Threonine accumulates in cells in a large amount in the later period of fermentation to form feedback inhibition, at the moment, the cell permeability needs to be changed to improve the secretion efficiency, the concentration of chitosan is 10,20,40,60,80 and 100, and the unit is mg/L, as shown in figure 4, when the concentration of chitosan is lower, the yield of threonine is positively correlated with the yield of threonine, but when the concentration of chitosan is too high, the yield of threonine is reduced, and when the concentration of chitosan is too high, the strain is obviously inhibited by excessive chitosan, the activity of the strain is lower, and the acid yield is correspondingly reduced.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. The production process of high-purity threonine comprises a fermentation process and an extraction and separation process.
2. The process according to claim 1, wherein the fermentation step comprises the steps of:
inoculating the L-threonine production strain seed liquid into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 1-2% for fermentation, controlling the stirring speed at 300-37 ℃ and the dissolved oxygen at 500rpm, controlling the dissolved oxygen at 15-20% by ventilation and stirring, defoaming by using a foam killer, and stopping fermentation when the fermentation time is 36h, and collecting the fermentation liquid.
3. The production process according to claims 1-2, wherein the extraction separation process comprises the steps of:
centrifuging the fermentation liquid for 3-5min at 4000-; filtering the threonine feed liquid by using a ceramic membrane, and collecting filtrate; performing primary decolorization treatment on the filtrate with activated carbon by plate-type closed filter for 30-90 min; performing secondary decolorization treatment on the decolorized clear liquid in a decolorizing membrane to obtain decolorized clear liquid; passing the decolorized clear liquid through an ultrafiltration membrane, and collecting ultrafiltrate; concentrating the ultrafiltrate to one fourth of the original volume, slowly cooling to 16 deg.C, and adjusting to obtain isoelectric solution; precipitating with isoelectric solution for 6-18 hr, centrifuging, collecting crystallized threonine, and oven drying.
4. The production process according to claim 2, wherein in the fermentation step,
the sugar content is controlled to be 2-3% by feeding 50% of sucrose solution until the fermentation is finished.
5. The production process according to claim 2, wherein in the fermentation step, the pH is controlled to 7.0 by feeding 20 to 30% ammonia water until the end of the fermentation.
6. The production process of claim 2, wherein in the fermentation step, the fermentation is carried out for about 20 hours, and hydrogen peroxide is fed into the fermentation tank at a flow rate of 2-3ml/h per liter of fermentation liquid until the fermentation is finished.
7. The process according to claim 2, wherein the fermentation step comprises fermenting for about 20 hours, and the mixed aqueous solution of succinic acid and sodium citrate is fed to the fermentation tank at a flow rate of 10-20ml/h per liter of fermentation broth until the fermentation is completed.
8. The production process according to claim 2, wherein in the fermentation step, chitosan is added to the fermentation tank at one time after the fermentation is carried out for about 30 hours, and the concentration of the chitosan is controlled to be 40-60 mg/L.
9. The process according to claim 2, wherein the fermentation medium comprises:
60g/L of sucrose, 30g/L of glucose, 20g/L of corn steep liquor, 5g/L of ammonium sulfate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, VB12mg/L，VH50μg/L。
10. The production process according to claim 7, wherein in the fermentation step, the concentrations of succinic acid and sodium citrate in the mixed aqueous solution are both 50 g/L.
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