CN110846350A - Threonine production and separation refining process - Google Patents
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Abstract
The invention belongs to the technical field of amino acid production, and discloses a threonine production and separation refining process, which comprises the following steps: step 1) preparing a fermentation culture medium, step 2) preparing threonine through fermentation, and step 3) separating and refining threonine. The process has high fermentation efficiency, and the high-purity threonine product is obtained by separation and refining.
Description
Technical Field
The invention belongs to the technical field of amino acid production, and particularly relates to a process for improving threonine fermentation efficiency.
Background
Threonine plays an increasingly important role in human life as one of amino acids essential to the human body. With the development of the breeding industry and the rapid increase of the livestock and poultry feed demand, threonine plays a role in the nutritional ingredients which must be taken from the outside and is more and more emphasized. Has wide application in medicine, food, feed and other fields. Threonine belongs to one of products of industrial fermentation, and according to data statistics, the global threonine supply in 2017 reaches 68.5 ten thousand tons, the equivalent increase is 15.5 percent, or the global threonine supply increases 9.2 ten thousand tons, and the increase is 80 percent from China. In 2017, the supply of Chinese threonine reaches 53.5 ten thousand tons, and the increase is 15.6 percent on the same scale, which accounts for 78 percent of the global market. China threonine production enterprises in 2017 mainly use plum blossom, Fufeng, Yipin and Chengfu, and supplement Dacheng and Xijie; international enterprises mainly use ajinomoto and ADM. In 2017, the domestic threonine is exported 37.4 ten thousand tons, accounting for 69.9 percent of the yield, and the domestic supply is 16.1 ten thousand tons, and the domestic demand is 13 ten thousand tons.
The production method of L-threonine includes protein hydrolysis, chemical synthesis and microbial fermentation. Because the protein hydrolysis method and the chemical synthesis method have the defects of complex process, low yield, large environmental pollution, high cost and the like, the method is difficult to be applied to industrial production and is basically not used any more. The microbial fermentation method has the characteristics of low production cost and small environmental pollution, and is a main method for industrially producing the L-threonine. Microbial fermentation refers to a process of converting a raw material into a target product through a specific metabolic pathway by using a microorganism under appropriate conditions. The production level of microbial fermentation depends mainly on the genetic characteristics of the species itself and the culture conditions. The strain is reformed by using modern genetic engineering technology, the biosynthesis of byproducts is reduced, and the gene expression of products is improved, so that the yield of the L-threonine is improved. With the development of genetic engineering technology and the increase of information content of industrial microorganisms, particularly the successful construction of an industrial biological vector system, researchers in the Soviet Union before the last 70 th century began to construct threonine engineering bacteria by using the genetic engineering technology, and reliable technical support is provided for screening excellent L-threonine producing bacteria and improving the acid production level of strains.
The microorganisms used for L-threonine fermentation are mainly of the genera Escherichia, Brevibacterium, Corynebacterium, Proteus, and the biosynthetic pathways in different microbial species are approximately the same. Escherichia coli is the main strain for producing threonine by microbial fermentation. The literature reports that in Escherichia coli, there are the glycolytic pathway (EMP), the tricarboxylic acid cycle (TCA), the pentose phosphate pathway (HMP), the salvage pathway, and the phosphotransferase system (PTS). The HMP pathway can provide a large amount of NADPH for amino acid synthesis, and has important significance. During the fermentation of L-threonine, glucose synthesizes oxaloacetate through glycolysis, the tricarboxylic acid cycle, and oxaloacetate is an important intermediate product and is an important precursor substance for L-threonine synthesis. Research shows that glyoxylate cycle does not appear in fermentation culture using glucose as a substrate in escherichia coli, which means that TCA cycle is the main oxidation mode in the fermentation process of escherichia coli; meanwhile, phosphoenolpyruvate carboxylase (PPC) -catalyzed reactions are the major anaplerotic reaction of the TCA cycle.
For the research on threonine fermentation culture medium, synthetic pathway and the like, the applicant's prior patent technology has been extensively elucidated, the research on the threonine synthesis mechanism is further carried out, and culture medium, culture conditions and the like are optimized, so as to further improve the fermentation efficiency of threonine and reduce the synthesis of metabolic byproducts.
The applicant's prior patent technology ' a process for improving threonine fermentation efficiency ', improves the fermentation efficiency by optimizing the culture medium and culture parameter steps, and on the basis of the technology, the applicant continues to separate and refine threonine in the fermentation liquor.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a threonine production and separation refining process.
The invention is realized by the following technical scheme:
a threonine production and separation refining process is characterized by comprising the following steps: step 1) preparing a fermentation culture medium, step 2) preparing threonine through fermentation, and step 3) separating and refining threonine.
Further, the components of the fermentation medium are as follows: 40-60g/L of glucose, 20-25g/L of corn steep liquor, 0.5-0.7g/L of monopotassium phosphate, 0.5-0.7g/L of dipotassium phosphate, 0.10-0.15g/L of magnesium sulfate, 50-100mg/L of N-methyl aspartic acid, 20-30mg/L of methionine, 10-15mg/L of ferrous sulfate heptahydrate, 10-15mg/L of manganese sulfate monohydrate, and the pH value is 6.5-7.0.
Further, the step 2) of preparing threonine by fermentation comprises the following steps: inoculating the threonine-producing Brevibacterium flavum seed solution into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 8-10% for fermentation, stopping fermentation when the fermentation time is 48-60h, and collecting the fermentation liquid.
Further, the step 3) of separating and purifying threonine comprises the following steps: centrifuging the fermentation liquid for 3-5min at 4500-; filtering with a microfiltration membrane, and collecting filtrate; ultrafiltering with ultrafiltration membrane, collecting ultrafiltrate, pumping into decolorizing tank for decolorizing treatment, adding powdered activated carbon 0.3-0.5% of the ultrafiltrate, controlling the temperature in the decolorizing tank at 45-50 deg.C, pH at 6.5-7.0, decolorizing for 20-30min, filtering with plate frame to remove activated carbon, collecting clear liquid, concentrating to one fourth of original volume, slowly cooling to 18 deg.C, adjusting to pH 6.1-6.2, and settling for 8-18 hr; centrifuging to collect wet threonine crystal, and drying the wet threonine crystal.
Preferably, the microfiltration membrane is an inorganic ceramic membrane, the cut-off molecular weight is 10000Da, and the microfiltration temperature is 40 ℃.
Preferably, the ultrafiltration membrane is a polyvinylidene fluoride ultrafiltration membrane, the molecular weight cutoff is 300Da, and the ultrafiltration temperature is 40 ℃.
Preferably, in the whole fermentation process, the sugar content in the fermentation liquid is controlled to be 0.5% by feeding the nutrient solution with the glucose concentration of 200-300g/L, and the pH of the fermentation liquid is controlled to be 6.5 by feeding ammonia water.
Preferably, the fermentation conditions are: the temperature is 30-32 ℃, the tank pressure is 0.03-0.04MPa, the ventilation volume is 0.5-0.6vvm, and the rotating speed is 50-100 rpm.
Preferably, the nutrient solution contains 3-5g/L of N-methyl aspartic acid and 1-2g/L of methionine.
More preferably, the nutrient solution contains 5g/L of N-methyl aspartic acid and 1g/L of methionine.
The starting point and the beneficial effects of the research of the invention mainly comprise but are not limited to the following aspects:
in the threonine synthesis mechanism, by increasing the intracellular concentration of methionine to increase the amount of homoserine, which is an intermediate product in the pathway of L-aspartate to threonine synthesis, the accumulation of S-adenosylmethionine, which is a metabolite of methionine, inhibits succinyl homoserine synthase, thereby promoting L-threonine synthesis;
n-methyl aspartic acid can improve methyl for threonine synthesis, and can also be used as an intermediate product for threonine synthesis, thereby improving L-threonine synthesis;
the N-methyl aspartic acid and the methionine carry out dual regulation and control on the threonine synthesis way, the synergistic performance is good, the acid production efficiency is improved, the fermentation time is shortened, and the cost is saved.
Experimental data show that the yield of threonine can be improved by both N-methyl aspartic acid and methionine, and the yield of threonine can be improved by more than 20 percent by the synergistic effect of N-methyl aspartic acid and methionine compared with the yield of threonine without adding N-methyl aspartic acid and methionine.
The invention has simple and feasible process, high yield and purity of threonine, and can reach the pharmaceutical or food grade standard.
Drawings
FIG. 1: the influence of N-methyl aspartic acid and methionine on the growth of thallus;
FIG. 2: the effect of N-methylaspartic acid and methionine on threonine production;
FIG. 3: influence of the addition of N-methylaspartic acid in the fermentation medium on the yield of threonine;
FIG. 4: influence of the amount of methionine added to the fermentation medium on the threonine production.
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
A threonine production and separation refining process comprises the following steps:
step 1) preparing a fermentation medium: 60g/L glucose, 20g/L corn steep liquor, 0.5g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 0.10g/L magnesium sulfate, 100mg/L N-methyl aspartic acid, 30mg/L methionine, 10mg/L ferrous sulfate heptahydrate, 10mg/L manganese sulfate monohydrate, and the pH value is 6.5-7.0;
step 2) preparing threonine by fermentation: inoculating Brevibacterium flavum ATCC14067 seed solution (the OD600 of the seed solution is 16) into a fermentation tank containing a fermentation medium according to the inoculation amount of 8 percent for fermentation, wherein the temperature is 32 ℃, the tank pressure is 0.04MPa, the ventilation volume is 0.5vvm, the rotating speed is 100rpm, the fermentation time is 60h, stopping fermentation, and collecting fermentation liquor; in the whole fermentation process; in the whole fermentation process, the sugar content in the fermentation liquor is controlled to be 0.5% by feeding nutrient solution with the glucose concentration of 300g/L, and the pH value of the fermentation liquor is controlled to be 6.5 by feeding ammonia water.
The nutrient solution contains 5g/L of N-methyl aspartic acid and 1g/L of methionine;
step 3) separation and purification of threonine: centrifuging the fermentation liquid for 3min at 5000rpm by using a disc centrifuge, and collecting the upper layer liquid; filtering with a microfiltration membrane, and collecting filtrate; performing ultrafiltration with ultrafiltration membrane, collecting ultrafiltrate, pumping into a decolorizing tank for decolorizing treatment, adding powdered activated carbon 0.5% of the ultrafiltrate, controlling the temperature at 45 deg.C, pH at 6.5, decolorizing for 30min, filtering with plate frame to remove activated carbon, collecting clear solution, concentrating to one fourth of original volume, slowly cooling to 18 deg.C, adjusting to pH 6.1, and settling for 12 hr; centrifugally collecting wet threonine crystals, and drying the wet crystals to obtain the threonine crystal;
the microfiltration membrane is an inorganic ceramic membrane, the cut-off molecular weight is 10000Da, and the microfiltration temperature is 40 ℃; the ultrafiltration membrane is a polyvinylidene fluoride ultrafiltration membrane, the molecular weight cutoff is 300Da, and the ultrafiltration temperature is 40 ℃.
The threonine product has 99.2% purity and high purity by HPLC detection, and can be used as threonine for medicine or food.
Example 2
A threonine production and separation refining process comprises the following steps:
step 1) preparing a fermentation medium: 50g/L glucose, 20g/L corn steep liquor, 0.7g/L potassium dihydrogen phosphate, 0.7g/L dipotassium hydrogen phosphate, 0.10g/L magnesium sulfate, 75mg/L N-methyl aspartic acid, 25mg/L methionine, 10mg/L ferrous sulfate heptahydrate, 10mg/L manganese sulfate monohydrate, and the pH value is 6.5-7.0;
step 2) preparing threonine by fermentation: inoculating Brevibacterium flavum ATCC14067 seed solution (the OD600 of the seed solution is 12) into a fermentation tank containing a fermentation medium according to the inoculation amount of 10 percent for fermentation, wherein the temperature is 30-32 ℃, the tank pressure is 0.03MPa, the ventilation volume is 0.6vvm, the rotating speed is 50rpm, the fermentation time is 50h, stopping fermentation, and collecting fermentation liquor; in the whole fermentation process; in the whole fermentation process, the sugar content in the fermentation liquor is controlled to be 0.5% by feeding nutrient solution with the glucose concentration of 200g/L, and the pH value of the fermentation liquor is controlled to be 6.5 by feeding ammonia water.
The nutrient solution contains 3g/L of N-methyl aspartic acid and 2g/L of methionine;
step 3) separation and purification of threonine: centrifuging the fermentation broth with a disk centrifuge at 4500rpm for 5min, and collecting the upper layer liquid; filtering with a microfiltration membrane, and collecting filtrate; performing ultrafiltration with ultrafiltration membrane, collecting ultrafiltrate, pumping into a decolorizing tank for decolorizing treatment, adding powdered activated carbon 0.4% of the ultrafiltrate, controlling the temperature at 45 deg.C, pH at 6.5, decolorizing for 20min, filtering with plate frame to remove activated carbon, collecting clear solution, concentrating to one fourth of original volume, slowly cooling to 18 deg.C, adjusting to pH 6.2, and settling for 8 hr; centrifugally collecting wet threonine crystals, and drying the wet crystals to obtain the threonine crystal;
the microfiltration membrane is an inorganic ceramic membrane, the cut-off molecular weight is 10000Da, and the microfiltration temperature is 40 ℃; the ultrafiltration membrane is a polyvinylidene fluoride ultrafiltration membrane, the molecular weight cutoff is 300Da, and the ultrafiltration temperature is 40 ℃.
The threonine product has 99.1% purity and high purity by HPLC detection, and can be used as threonine for medicine or food.
Example 3
A threonine production and separation refining process comprises the following steps:
step 1) preparing a fermentation medium: 50g/L glucose, 25g/L corn steep liquor, 0.5g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 0.10g/L magnesium sulfate, 50mg/L N-methyl aspartic acid, 20mg/L methionine, 10mg/L ferrous sulfate heptahydrate, 10mg/L manganese sulfate monohydrate, and the pH value is 6.5-7.0;
step 2) preparing threonine by fermentation: inoculating Brevibacterium flavum ATCC14067 seed solution (the OD600 of the seed solution is 12) into a fermentation tank containing a fermentation medium according to the inoculation amount of 9 percent for fermentation, wherein the temperature is 30 ℃, the tank pressure is 0.03MPa, the ventilation volume is 0.6vvm, the rotating speed is 70rpm, the fermentation time is 54h, stopping fermentation, and collecting fermentation liquor; in the whole fermentation process; in the whole fermentation process, the sugar content in the fermentation liquor is controlled to be 0.5% by feeding nutrient solution with the glucose concentration of 300g/L, and the pH value of the fermentation liquor is controlled to be 6.5 by feeding ammonia water.
The nutrient solution contains 4g/L of N-methyl aspartic acid and 2g/L of methionine;
step 3) separation and purification of threonine: centrifuging the fermentation liquid for 4min at 5000rpm by using a disc centrifuge, and collecting the upper layer liquid; filtering with a microfiltration membrane, and collecting filtrate; performing ultrafiltration by using an ultrafiltration membrane, collecting ultrafiltrate, pumping the ultrafiltrate into a decolorizing tank for decolorizing, adding powdered activated carbon accounting for 0.3 percent of the mass of the ultrafiltrate into the decolorizing tank, controlling the temperature in the decolorizing tank to be 45 ℃, controlling the pH value to be 6.5, decolorizing for 30min, filtering the activated carbon by using a plate frame, collecting clear liquid, concentrating the clear liquid to be one fourth of the original volume, then entering an isoelectric tank, slowly cooling to 18 ℃, adjusting to be an isoelectric solution with the pH value of 6.2, and settling for 18 hours; centrifugally collecting wet threonine crystals, and drying the wet crystals to obtain the threonine crystal;
the microfiltration membrane is an inorganic ceramic membrane, the cut-off molecular weight is 10000Da, and the microfiltration temperature is 40 ℃; the ultrafiltration membrane is a polyvinylidene fluoride ultrafiltration membrane, the molecular weight cutoff is 300Da, and the ultrafiltration temperature is 40 ℃.
Example 4
And (4) analyzing and measuring methods.
Measurement of cell concentration: and measuring the OD value of the thallus by adopting an ultraviolet-visible spectrophotometer under the condition of 600 nm wavelength.
Determination of threonine in fermentation broth: and extracting fermentation liquor at different time points, centrifuging, and measuring the yield of the threonine by using a supernatant and a paper chromatography.
The effects of mono, N-methylaspartic acid and methionine on the growth of the bacteria.
Comparison 1: the fermentation medium and the nutrient solution are not added with N-methyl aspartic acid and methionine, and the rest is the same as the example 1;
comparison 2: the fermentation medium and the nutrient solution are not added with N-methyl aspartic acid, and the rest is the same as the example 1;
comparison 3: the fermentation medium and the nutrient solution are not added with methionine, and the rest is the same as the example 1;
experimental groups: example 1.
Setting different time points to detect OD of thallus600The values and time points are respectively selected to be 0,12 and 24,36,48,60,72。
As shown in FIG. 1, the transverse data analysis showed that the cell concentration increased rapidly with the increase of fermentation time, reaching a maximum value of 57.9 (OD) after 36h600Value), then the concentration of the thalli is not obviously increased and is maintained in a stable state, and when the fermentation lasts for 72 hours, the concentration of the thalli is obviously reduced, which may be the cause of the deterioration of the culture environment; longitudinally observing that the concentration of thalli of each group is not obviously different in the early stage of fermentation (within 24 h), along with the increase of fermentation time, the concentration of thalli of an experimental group is higher than that of comparison 1 and comparison 3, and is closer to that of comparison 2, by researching the peak value of the concentration of thalli of each group, the comparison 1 is the lowest, and the comparison 3 is the next, and the comparison 2 is not obviously different from the experimental group; in conclusion, N-methyl aspartic acid has no obvious change on the growth of thalli, and methionine can improve the growth rate of thalli to a certain extent.
The effect of di, N-methylaspartic acid and methionine on threonine production.
Comparison 1: the fermentation medium and the nutrient solution are not added with N-methyl aspartic acid and methionine, and the rest is the same as the example 1;
comparison 2: the fermentation medium and the nutrient solution are not added with N-methyl aspartic acid, and the rest is the same as the example 1;
comparison 3: the fermentation medium and the nutrient solution are not added with methionine, and the rest is the same as the example 1;
experimental groups: example 1.
Threonine production (g/L) was measured by setting different time points, 0,12,24,36,48,60,72, respectively.
As shown in fig. 2, in the initial stage of fermentation, the threonine content increases slowly with the increase of fermentation time, and in the middle stage of fermentation, the thallus concentration is relatively high and is in a relatively stable stage, at this time, the threonine yield increases significantly, and reaches a peak value after about 60 hours; longitudinal data analysis, the initial stage of fermentation, because threonine output of each group all is in the low level, the output difference of each group is not big, the middle and later stages of fermentation, each group all has obvious promotion, through the peak value output discovery of each group of contrast, contrast 1 is minimum, next contrast 2, contrast 3 again, the highest is the experimental group, show, N-methyl aspartic acid and methionine all can promote the output of threonine, the two synergism compares with contrast 1 that does not add N-methyl aspartic acid and methionine, 23 percentage points have been promoted.
And thirdly, influence of the addition amount of the N-methyl aspartic acid and the methionine in the fermentation medium on the yield of the threonine.
The concentration gradient of N-methyl aspartic acid is set as: 0,25,50,75,100,125,150 (mg/L), detecting the maximum yield of the threonine under each concentration gradient, and drawing a curve, wherein N-methyl aspartic acid has a certain positive regulation effect on the yield of the threonine, and the addition amount of 50-100mg/L is most suitable in consideration of cost and other factors, as shown in figure 3.
The concentration gradient of methionine was set as: 0,10,20,30,40,50 and 60 (mg/L), detecting the maximum yield of the threonine under each concentration gradient, and drawing a curve, wherein as shown in figure 4, the methionine can regulate the yield of the threonine fermented by the brevibacterium flavum, and the addition amount of 20-30mg/L is most suitable.
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. A threonine production and separation refining process is characterized by comprising the following steps: step 1) preparing a fermentation culture medium, step 2) preparing threonine through fermentation, and step 3) separating and refining threonine.
2. The process of claim 1, wherein the fermentation medium comprises: 40-60g/L of glucose, 20-25g/L of corn steep liquor, 0.5-0.7g/L of monopotassium phosphate, 0.5-0.7g/L of dipotassium phosphate, 0.10-0.15g/L of magnesium sulfate, 50-100mg/L of N-methyl aspartic acid, 20-30mg/L of methionine, 10-15mg/L of ferrous sulfate heptahydrate, 10-15mg/L of manganese sulfate monohydrate, and the pH value of the mixture is 6.5-7.0.
3. The process according to claim 1 or 2, wherein step 2) fermentative preparation of threonine comprises the following steps: inoculating the threonine-producing Brevibacterium flavum seed solution into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 8-10% for fermentation, stopping fermentation when the fermentation time is 48-60h, and collecting the fermentation liquid.
4. The process according to claim 3, wherein the step 3) of separating the purified threonine comprises the steps of: centrifuging the fermentation liquid for 3-5min at 4500-; filtering with a microfiltration membrane, and collecting filtrate; ultrafiltering with ultrafiltration membrane, collecting ultrafiltrate, pumping into decolorizing tank for decolorizing treatment, adding powdered activated carbon 0.3-0.5% of the ultrafiltrate, controlling the temperature in the decolorizing tank at 45-50 deg.C, pH at 6.5-7.0, decolorizing for 20-30min, filtering with plate frame to remove activated carbon, collecting clear liquid, concentrating to one fourth of original volume, slowly cooling to 18 deg.C, adjusting to pH 6.1-6.2, and settling for 8-18 hr; centrifuging to collect wet threonine crystal, and drying the wet threonine crystal.
5. The process according to claim 4, wherein the microfiltration membrane is an inorganic ceramic membrane, the molecular weight cut-off is 10000Da, and the microfiltration temperature is 40 ℃.
6. The process of claim 4, wherein the ultrafiltration membrane is a polyvinylidene fluoride ultrafiltration membrane with a molecular weight cut-off of 300Da and an ultrafiltration temperature of 40 ℃.
7. The process as claimed in claim 3, wherein the sugar content in the fermentation broth is controlled to 0.5% by feeding a nutrient solution having a glucose concentration of 200-300g/L and the pH of the fermentation broth is controlled to 6.5 by feeding ammonia water throughout the fermentation process.
8. The process according to claim 3, wherein the fermentation conditions are: the temperature is 30-32 ℃, the tank pressure is 0.03-0.04MPa, the ventilation volume is 0.5-0.6vvm, and the rotating speed is 50-100 rpm.
9. The process according to claim 7, wherein the nutrient solution contains 3-5g/L of N-methyl aspartic acid and 1-2g/L of methionine.
10. The process according to claim 9, wherein the nutrient solution comprises 5g/L N-methyl aspartic acid and 1g/L methionine.
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