阅读说明：本技术 一种苏氨酸生产和分离精制工艺 (Threonine production and separation refining process ) 是由 李德衡 赵兰坤 刘元涛 王小平 于 2019-12-09 设计创作，主要内容包括：本发明属于氨基酸生产技术领域,公开了一种苏氨酸生产和分离精制工艺,其包括如下步骤：步骤1)配置发酵培养基,步骤2)发酵制备苏氨酸,步骤3)分离精制苏氨酸。本发明工艺发酵效率高,通过分离精制得到高纯度的苏氨酸产品。(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.)

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.

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.