Method for producing 6s-5 methyltetrahydrofolic acid by using protoplast immobilization technology

Abstract

According to the method, engineering microorganisms are immobilized on stationary phases such as gelatin by applying a microorganism immobilization technology, cell walls of the microorganisms are removed by applying lysozyme, and 6s-5 methyltetrahydrofolic acid in cells can be easily extracted through cell membranes under the action of a surfactant. A culture solution and an eluent are made into mobile phases, the mobile phases flow through a stationary phase containing engineering bacteria, so that the 6s-5 methyltetrahydrofolic acid is continuously extracted from microorganisms, and after the 6s-5 methyltetrahydrofolic acid is collected, the treated mobile phases such as the culture solution and the eluent are circularly input into the stationary phase to form a continuous flow production process. The continuous flow process shows a milk squeezing effect, and the concentration balance of 6s-5 methyltetrahydrofolic acid in microorganisms is broken, so that the 6s-5 methyltetrahydrofolic acid is continuously biosynthesized and output. The technical problem of low concentration of biological fermentation final products at present is solved.

Description

1. Technical Field

[0001] The present invention belongs to the field of bio-fermentation technology of biomedicine and food additives, and specifically relates to a green process for fixing engineering microorganisms and preparing 6S-5-methyltetrahydrofolate by bio-fermentation. This method uses the engineering microorganism fixing technology and the technical condition of removing the microbial cell wall by lysozyme, so that the process realizes continuous flow circulation production, improves the utilization efficiency of production materials and the collection efficiency of products. 2. Background Technology

[0002] With the deepening of research on the biological activity of folic acid, 6s-5-methyltetrahydrofolate is the active form of folic acid in the human body, and 6s-5-methyltetrahydrofolate calcium is the most stable salt form of 6s-5-methyltetrahydrofolate known so far. It has the characteristics of easy absorption, high biological activity and good safety, and its market demand continues to grow. 6s-5-methyltetrahydrofolate calcium is increasingly widely used in the medical field, especially in preventing neural tube defects in newborns, improving cardiovascular health in adults, and assisting tumor treatment. With the aging of the population and the increase in the incidence of chronic diseases, its demand for clinical treatment and health care continues to grow. At present, 6s-5-methyltetrahydrofolate calcium is mainly produced by chemical synthesis in the industry. The existing chemical synthesis technology has the following three major defects:

[0003] 1. Traditional chemical synthesis requires the use of heavy metal catalysts, resulting in excessive product residues (>50ppm);

[0004] 2. The splitting process causes a yield loss of more than 70%;

[0005] 3. The cost of three wastes treatment accounts for more than 40% of the total cost.

[0006] Since the traditional chemical synthesis process requires chemical splitting of the final product racemate, the actual yield of the product is about 30%. Therefore, the synthesis of the initial raw material folic acid has a greater impact on the overall production cost. The synthesis of folic acid requires the participation of trichloroacetone, and the production of trichloroacetone requires a large amount of chlorine as a raw material, which makes the production of folic acid environmentally friendly. This has also led to the current increase in the price of folic acid raw materials, which has further increased the production cost of 6s-5-methyltetrahydrofolate.

[0007] Therefore, it is particularly important to find a method for producing 6S-5-methyltetrahydrofolate by bio-fermentation with organic matter as culture medium and genetically recombinant microorganisms, so as to achieve the goal of green environmental protection and reduce production costs. According to previous literature reports, the bottleneck of the current microbial fermentation technology for producing 6S-5-methyltetrahydrofolate is that the final product concentration is extremely low, with a maximum of about 300ug / L. The transformation of the "one-carbon unit" metabolic pathway in the engineered microorganisms E. coli, B. subtilis and L. lactis through genetic recombination technology in order to achieve the purpose of increasing the expression of 6S-5-methyltetrahydrofolate seems to have little effect. This makes it difficult to apply the bio-fermentation process in this field to large-scale production.

[0008] The reasons for the low content of the final product are mainly the following two aspects: First, in the biochemical pathway in the microorganism, 6s-5-methyltetrahydrofolate is one of the products in its "one-carbon metabolism" pathway. Its intermediates and itself have feedback inhibition in the metabolic pathway. When it accumulates to a certain concentration in the microorganism, it will remain stable. This is also a self-protection mechanism evolved by organisms. Second, as a methyl provider in the biochemical reaction, 6s-5-methyltetrahydrofolate generates methionine with homocysteine ​​in the microorganism and enters the "one-carbon metabolism cycle", so that its own content is constantly consumed, so it will not continue to accumulate in the microorganism. It can be seen that how to remove and collect 6s-5-methyltetrahydrofolate from the microorganism in a timely manner is the key to solving the current technical bottleneck.

[0009] This method uses microbial fixation technology to fix the engineered microorganisms on a stationary phase such as gelatin, and uses lysozyme to remove the cell wall of the microorganism. Under the action of the surfactant, the 6S-5-methyltetrahydrofolate in the cell can be easily extracted through the cell membrane. The culture fluid and the eluate are made into mobile phases, which flow through the stationary phase containing the engineered bacteria, so that the 6S-5-methyltetrahydrofolate is continuously extracted from the microorganism. After collecting the 6S-5-methyltetrahydrofolate, the treated culture fluid and eluate and other mobile phases are recycled and input into the stationary phase to form a continuous flow production process. This continuous flow process presents a "milking effect", breaking the concentration balance of 6S-5-methyltetrahydrofolate in the microorganism, allowing it to be continuously biosynthesized and output. It solves the technical problem of low concentration of the final product of biological fermentation that has been plaguing the current process. III. Summary of the invention

[0010] 1. Technical solution

[0011] Construction and expression of genetically recombinant microorganisms

[0012] The human MTHFR gene protein sequence was selected through the UniProt database, cloned and assembled into the host cell Escherichia coli (Eschericha coli DE3) for expression, and then through directed evolution and rational transformation processes, the engineered bacteria that efficiently expressed 6S-5-methyltetrahydrofolate were screened out as the strain for culture and subculture.

[0013] Take 5 ml of the culture solution containing the bacterial strain and add it to 100 ml of TB medium (containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 60 g / L glucose, 12.5 g / L dipotassium hydrogen phosphate and 2.5 g / L potassium dihydrogen phosphate, etc.), and induce culture on a shaking table at a temperature of 36.5°C and a pH value of 7.5 for 24 hours until the OD600nm value is 0.8, and refrigerate at 2-8°C for later use.

[0014] Immobilization technology of engineered microorganisms

[0015] Heat sterilize 1L of 500mg gelatin suspension, cool to 35℃, add 100ml of solution containing bacteria after induction culture, stir and mix well, pour into a container and refrigerate in a freezer at 2-8℃ for 2 hours, take out, soak in 100ml 30% glutaraldehyde saline for 2-3 hours, and use a grinder and a granulator to make the gel blocks into 1-2mm particles. Place the above gel particles in 100ml 45% glutaraldehyde saline solution and let stand for 2 hours, filter out the particles, rinse with saline and load into the stationary phase column for use.

[0016] The core technology of 6S-5-methyltetrahydrofolate biological expression

[0017] 100 ml of physiological saline solution containing 50 mg / L lysozyme (Lysozyme is derived from egg white) is circulated and flushed on the stationary phase containing the fixed microorganism gel for 20-30 minutes;

[0018] The culture fluid (containing 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, 60g / L glucose, 12.5g / L potassium dihydrogen phosphate and 2.5g / L potassium dihydrogen phosphate, 100ug / L ampicillin, 0.5mg / L Tween 80, 25% glycerol, etc.) is prepared into 2000ml mobile phase infusion stationary phase with physiological saline as solvent, the pH value is adjusted to 7.3, the temperature is maintained at 37.5°C, and the flow rate is controlled to 50ml / min. The outflowing mobile phase collection liquid is ultrafiltered by pressurization (the ultrafiltration membrane is a hollow fiber containing acetate fiber polymer material). The filtrate containing small molecular substances is stored in a closed container containing nitrogen for standby use. The culture fluid containing large molecular substances that cannot pass through the ultrafiltration membrane is refluxed to the mobile phase and mixed with it, and then re-input into the stationary phase for cyclic production. This process can be optimized to alternately use culture medium and pure water as the mobile phase to flush the stationary phase according to the concentration of the product extracted from the cell. However, it should be noted that pure water is a hypotonic substance for the substances in the protoplasm cells. Although it can provide osmotic pressure and is conducive to the overflow of intracellular substances, it can also easily cause the cells to absorb too much water, causing the cells to rupture and die. Therefore, it is particularly important to control the concentration and action time of the hypotonic eluent.

[0019] The filtrate was flushed with nitrogen and the pH value was adjusted to 3.0 with 0.2 mol / L hydrochloric acid to obtain a white solid precipitate. After filtration, water washing, ether elution and drying, 6s-5-methyltetrahydrofolate was obtained. The separated solution was adjusted to pH 7.5 with sodium hydroxide and mixed with the mobile phase to participate in the circulation production.

[0020] Finally, 6s-5-methyltetrahydrofolate with an optical purity of 99.9% is obtained, and the yield can reach 95.5%.

[0021] 2. Innovation

[0022] A continuous flow production system of 6S-5-methyltetrahydrofolate was constructed using protoplasm fixation technology, which enables the recycling of culture fluid and engineered bacteria in the system, saving material loss and thus reducing costs;

[0023] A dynamic separation and collection mode for the product 6s-5-methyltetrahydrofolate was developed, which enables the engineered microorganisms to continuously biochemically synthesize 6s-5-methyltetrahydrofolate, solving the technical problems of low final product concentration, great difficulty in extraction and separation, and high cost in the current biological fermentation process;

[0024] Ultrafiltration membrane is used to achieve macromolecular separation (residue < 0.01%), improving separation efficiency and purity of the final product;

[0025] The realization of continuous flow dynamic production technology improves the efficiency of equipment use and the smoothness and continuity of the production process, providing support for automation design.

[0026] 3. Technical Effect

[0027] Compared with the previous fermentation method, the present invention has obvious advantages: the production materials of the traditional fermentation method: the culture solution and the engineered microorganism can only be used once and cannot be recycled; in addition, the most critical thing is that the concentration of 6s-5-methyltetrahydrofolate in the final product is extremely low, only about 300 mg / L at most, which cannot meet the requirements of large-scale production. The method of the present invention can realize the continuous production of recycled production materials, and the collection of the final product realizes the process of dynamic accumulation. Calculated based on the production cycle of a batch of 3 days, the cumulative product concentration can reach 70g / L, which is 233 times that of the traditional fermentation process, thus meeting the conditions for large-scale industrial production of the process.

[0028] The specific advantages are compared in the table below.

[0029] index Traditional fermentation process The present invention Production efficiency 300mg / L per batch 70g / L per batch Production costs 10000 Yuan / kg 1000 Yuan / kg Production Fluency Intermittent continuous Material Usage One-time Recycling Separation and extraction The procedure is complicated and there are many impurities Simple, less impurities 4. Specific implementation methods

[0030] Example 1

[0031] Under the conditions of pH 7.0 and 37°C, 2000 ml of the prepared culture medium (containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 60 g / L glucose, 12.5 g / L potassium dihydrogen phosphate and 2.5 g / L potassium dihydrogen phosphate, 100 ug / L ampicillin, 0.5 mg / L Tween 80, 25% glycerol, etc.) was used as the mobile phase and passed through the stationary phase filled with fixed protoplasm gelatin particles at a flow rate of 50 ml / min.

[0032] Proceed in sequence:

[0033] 1. Ultrafilter the mobile phase (the filter membrane contains hollow fibers made of cellulose acetate polymer);

[0034] 2. The pH value of the filtrate is adjusted to 3.0 to generate a white solid precipitate, which is then filtered, washed with acetone, and dried;

[0035] 3. The separated solution is adjusted to pH 7 with NaOH and then mixed with the mobile phase for recycling.

[0036] Obtain the product optical rotation [α]D 25 =+18.5°, purity>95.8% as determined by HPLC.

[0037] Example 2

[0038] Under pH 7.5 and 37.5°C conditions, 2000ml of the prepared culture solution (containing 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, 60g / L glucose, 12.5g / L potassium dihydrogen phosphate and 2.5g / L potassium dihydrogen phosphate, 100ug / L ampicillin, 0.5mg / L Tween 80, 25% glycerol, etc.) was used as the mobile phase. First, the valve of the liquid preparation port of the stationary phase filling column containing fixed protoplasm gelatin particles was closed, and then after the mobile phase was filled with the stationary phase and mixed evenly, the mobile phase input switch was closed, and the column was allowed to stand for 4-6 hours, the temperature was maintained at 37.5°C, and the pH value was adjusted to 7.5. The culture medium was drained for standby use (infused back into the culture medium storage tank), and 2000 ml of eluent (0.9% saline, 100 g / L glucose, 60% glycerol, 5 mg / L Tween 80) was infused into the stationary phase at a flow rate of 50 ml / min.

[0039] Proceed in sequence:

[0040] 1. Ultrafiltration of the mobile phase (culture solution or eluent) (the filter membrane contains hollow fibers made of cellulose acetate polymer);

[0041] 2. The pH value of the filtrate is adjusted to 2.8 to generate a white solid precipitate, which is then filtered, washed with alcohol, and dried;

[0042] 3. The separated solution is adjusted to pH 7 with NaOH and then mixed with the mobile phase for recycling.

[0043] Obtain the product optical rotation [α]D 25 =+18.5°, purity>96.5% as determined by HPLC.

[0044] Example 3

[0045] Under the conditions of pH 7.8 and 38°C, 2000ml of the prepared culture solution (containing 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, 60g / L glucose, 12.5g / L potassium dihydrogen phosphate and 2.5g / L potassium dihydrogen phosphate, 100ug / L ampicillin, 0.5mg / L Tween 80, 25% glycerol, etc.) was used as the mobile phase. The valve of the liquid dispensing port of the stationary phase filling column containing the fixed protoplasm gelatin particles was first closed, and then after the mobile phase was fully infused with the stationary phase and mixed evenly, the mobile phase input switch was closed, and the mobile phase was left standing for 4-6 hours, the temperature was maintained at 37.5°C, and the pH value was adjusted to 7.5. The culture solution was discharged for standby use (infused back into the culture solution storage tank), and the stationary phase was infused with 2000ml of eluent (pure water), and the stationary phase was passed through at a flow rate of 100ml / min. After washing the stationary phase with the eluent for 3 hours, the eluent was used to wash the stationary phase for 3 hours, and the two were performed alternately.

[0046] Proceed in sequence:

[0047] 1. Ultrafiltration of the mobile phase (culture solution or eluent) (the filter membrane contains hollow fibers made of cellulose acetate polymer);

[0048] 2. The pH value of the filtrate is adjusted to 3.2 to generate a white solid precipitate, which is then filtered, rinsed with petroleum ether, and dried;

[0049] 3. The separated solution is adjusted to pH 7 with NaOH and then mixed with the mobile phase (culture solution and eluent are infused into their respective storage tanks) for recycling.

[0050] Obtain the product optical rotation [α]D 25 =+18.5°, purity>98.2% as determined by HPLC. V. Description of the Figures

[0052] Figure 1 Schematic diagram of process equipment.

Claims

1. A method for producing 6S-5-methyltetrahydrofolate by protoplasm fixation technology, characterized in that include: Processes such as removal of microbial cell walls, microbial fixation, preparation of culture medium mobile phase and stationary phase containing protoplasm fixation technology, and ultrafiltration separation.

2. The present invention establishes a continuous flow production process for dynamic extraction, separation and collection of substances in microbial cells, characterized in that The device has the advantages of continuous collection and recycling, and continuously improves the collection efficiency of 6S-5-methyltetrahydrofolate.

3. The method according to claim 1, characterized in that The culture solution is prepared as follows: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 60 g / L glucose, 12.5 g / L dipotassium hydrogen phosphate and 2.5 g / L potassium dihydrogen phosphate, 100 ug / L ampicillin, 0.5 mg / L Tween 80, and 25% glycerol aqueous solution.

4. The method according to claim 1, characterized in that The induction and reaction process conditions are controlled within the range of pH 6.8-8.0 and temperature 36-40°C.

5. The method according to claim 2, characterized in that The recycling mode of reaction culture medium and eluent realizes the optimization of production cost and dynamic extraction and collection of products by means of reflux recycling of mobile phase.

6. The method according to claim 2, characterized in that The eluent has a formula consisting of an aqueous solution of 0.9% saline, 100 g / L glucose, 60% glycerol, and 5 mg / L Tween 80.

7. The method according to claim 2, characterized in that An extraction method in which the culture medium and the eluent are used as the mobile phase for the stationary phase alternately (the culture medium is used for washing for 3-6 hours, and then the eluent is used for washing for 2-3 hours).

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