Method for producing L-5 methyltetrahydrofolic acid based on metal organic framework material (MOFs) immobilized protoplast

Abstract

The invention discloses a 5-MTHF production method based on MOFs immobilized protoplast, which realizes efficient directional release of intracellular products by replacing engineering bacteria cell walls with MOFs and combining a gradient solvent extraction technology. The method has the advantages of high recovery rate (more than 85%), excellent product purity (more than 98%), recyclability of the MOFs material and the like, and is suitable for large-scale production of active folic acid in medicine and food industries.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a method for efficiently producing L-5-methyltetrahydrofolic acid (5-MTHF) by using metal-organic framework (MOFs) materials to replace the cell wall of engineering bacteria and through immobilized protoplasts and gradient solvent extraction technology. Technical Background

[0002] 1. Existing Technical Problems

[0003] L-5-methyltetrahydrofolic acid is the active form of folic acid. At present, the more promising production method is microbial fermentation, but it has the following defects:

[0004] The cell wall hinders the release of intracellular products. Traditional cell wall breaking methods (ultrasonic, enzymatic hydrolysis) are prone to damage the product structure, resulting in low recovery rate (<50%);

[0005] During solvent extraction, it is necessary to break the cell wall repeatedly, the operation is complex and impurities are easily introduced, affecting the product purity (<90%); The engineering bacteria can only be used once and cannot be reused, resulting in low production efficiency and high cost.

[0006] 2. Related Technical Progress

[0007] The cell wall of traditional Escherichia coli is composed of a peptidoglycan layer, but its mechanical strength is limited and it is easily affected by environmental stress, and it has a very large hindrance in the extraction of active substances in the cytoplasm during bioprocessing. Therefore, it is particularly important to explore a new engineering bacteria extraction process that can break through the hindrance of the engineering bacteria cell wall and maintain the continuous output of the target substance in the living state of the bacteria!

[0008] There is a literature report (J. Biotechnol, 2020) on the production of intracellular products by using protoplast fermentation with the application of protoplast fixation technology, but there are problems of poor stability and easy lysis. There are also reports on the fixation of Escherichia coli with the cell wall removed by using inorganic porous materials such as activated carbon and calcium alginate, but their skeleton materials have poor biocompatibility and are easily detached from the cells, resulting in the death of the bacteria; moreover, the specific surface area of the inorganic porous materials is not enough to carry enough living engineering bacteria, making the biorecovery process ineffective.

[0009] In recent years, MOFs have been tried for microbial interface modification due to their high specific surface area, adjustable pore size and functional diversity, but the existing technologies have the following problems:

[0010] (1), The binding between MOFs and the bacterial interface is not tight and is easy to fall off;

[0011] (2), After material loading, it hinders transmembrane material exchange and affects the extraction of the target product;

[0012] (3), Single function and lack of dynamic responsiveness.

[0013] MOF materials have been applied in the field of biological immobilization. Existing inventions have disclosed methods for coating enzymes with MOFs, but do not involve the replacement of microbial cell walls. The present invention utilizes the negative charges and abundant organic functional groups (such as phospholipids, membrane proteins, etc.) on the surface of Escherichia coli cell membranes to attract the positive charges in the metal ion clusters of MOF precursors. A MOF layer is in-situ grown by a mild solvothermal method. This method can form an "armor" structure that uniformly coats the surface of Escherichia coli cell membranes.

[0014] 3. Necessity of the invention

[0015] There is an urgent need to develop a 5-MTHF fermentation production process method for engineering bacteria that combines efficient product release, stable cell structure, high biocompatibility, and reusability. Summary of the invention

[0016] Object of the invention

[0017] To provide a method for highly efficient and high-purity production of 5-MTHF by immobilizing protoplasts with MOF artificial cell walls and combining gradient solvent extraction technology.

[0018] Technical solution

[0019] 1. Construction and cultivation of engineering bacteria

[0020] Genetically engineered Escherichia coli is selected, overexpressing folic acid synthesis genes (folE, folP, metF), and knocking out the methyltransferase gene (metE);

[0021] A fed-batch fermentation process is adopted, controlling the carbon-nitrogen ratio (C / N = 10:1 - 20:1) and dissolved oxygen (DO ≥ 30%), and the cell density reaches OD600 = 60 - 80.

[0022] 2. Preparation of protoplasts and MOF coating

[0023] Cell wall removal: The cells are suspended in an isotonic buffer (0.5 M sucrose, pH 7.0) containing lysozyme (1 - 5 mg / mL), treated at 37 °C for 30 - 60 minutes, and centrifuged to obtain protoplasts;

[0024] In-situ synthesis of MOFs: The protoplasts are mixed with MOF precursors (molar ratio of 2-methylimidazole to zinc nitrate 4:1), and by co-incubating with Escherichia coli with the cell wall removed, a ZIF-8 layer with a thickness of 50 - 200 nm and a pore size of 0.5 - 1.2 nm is formed on the surface of the protoplasts in the form of a biomimetic mineralization body;

[0025] Functional modification: Graft amino groups (-NH2) or carboxyl groups (-COOH) on the surface of ZIF-8 to enhance the affinity adsorption ability for 5-MTHF.

[0026] 3. Gradient solvent extraction

[0027] Primary extraction: Add ethyl acetate (volume ratio 1:1), shake for 30 minutes, penetrate the pores of MOFs to dissolve cell membrane lipids, and release free 5-MTHF;

[0028] Secondary extraction: Replace it with a methanol-water mixed solvent (volume ratio 7:3, containing 0.1 M EDTA), adjust the pH to 4.0, and dissociate 5-MTHF bound to proteins;

[0029] MOFs responsive release: Trigger the disintegration of the MOFs structure by adding 0.1 M citric acid to release the encapsulated metal ions (Zn 2 +), promoting the complete dissociation of the product.

[0030] 4. Product purification and MOFs regeneration

[0031] The extract is passed through a 0.22 μm ultrafiltration membrane to remove MOFs fragments, and then purified by an anion exchange resin (Dowex1×2) and eluted with a methanol gradient;

[0032] The MOFs fragments can be washed with ethanol and dried in vacuo, and can be reused 3 - 5 times.

[0033] The technical advantages of this invention are reflected in the following four aspects:

[0034] 1. High efficiency: The artificial cell wall of MOFs provides a directional diffusion channel, and combined with gradient extraction, the recovery rate of 5-MTHF is increased to over 85% (traditional method < 50%);

[0035] 2. High purity: The pore size of MOFs selectively excludes macromolecular impurities, and the product purity > 98%;

[0036] 3. Low cost: The MOFs material can be recycled, reducing the production cost by 30% - 40%;

[0037] 4. Environmental protection: Avoid breaking the cell wall with strong acids / alkalis and reduce waste emissions. Brief description of the drawings

[0038] Figure 1 : Schematic diagram of the structure of MOFs-coated protoplasts;

[0039] Figure 2 : Process flow chart of gradient solvent extraction;

[0040] Figure 3 : HPLC detection chromatogram of 5-MTHF calcium salt in the examples. Detailed implementation manners

[0041] Example 1: Preparation of MOFs-Coated Protoplasts

[0042] 1. Ferment 200 ml of genetically engineered E. coli BL21 (containing plasmid pET28a-folE-metF) in TB medium until OD600 = 60;

[0043] 2. Centrifuge to collect the cells, treat with lysozyme buffer for 45 minutes to obtain 100 ml of protoplast suspension solution for later use;

[0044] 3. Preparation of ZIF-8: Dissolve 5 g of Zn(NO3) 2. 6H2O in 150 ml of methanol solution to form solution A, and dissolve 6 g of 2-methylimidazole in 150 ml of methanol solution to form solution B. Subsequently, under ultrasonic conditions, add solution A dropwise to solution B, continue ultrasonic treatment for 15 minutes, then centrifuge, and redisperse the obtained solid in 150 ml of methanol to obtain solution C. Mix solution C with 5 g of Zn(NO3) 2. 6H2O, place it in a 500 ml reaction kettle, react at 120 °C for 2 hours, naturally cool to room temperature, centrifuge, wash with methanol multiple times, and vacuum dry the product to obtain 15 g of solid powder.

[0045] 4. Slowly add 15 g of ZIF-8 precursor (2-methylimidazole: zinc nitrate = 4:1) solid powder to a reaction kettle containing 100 ml of protoplast suspension, continuously stir and mix evenly, react at 25 °C for 2 hours, and centrifuge to obtain MOFs-coated protoplast particles.

[0046] Example 2: Gradient Solvent Extraction of 5-MTHF

[0047] 1. Mix the immobilized protoplasts with ethyl acetate (volume ratio 1:1), shake and extract at 30 °C for 30 minutes, and centrifuge to collect the supernatant (containing free 5-MTHF);

[0048] 2. Add methanol-water (7:3) solution to the remaining precipitate, adjust the pH to 4.0, add 0.1 M citric acid, react at 40 °C for 1 hour, and centrifuge to combine the extraction solutions;

[0049] 3. After ultrafiltration and purification, the purity of 5-MTHF detected by HPLC reaches 98.7%, and the total recovery rate is 86.2%.

Claims

1. A method for producing L-5-methyltetrahydrofolic acid, characterized in that It includes the following steps: Fermentation of engineered bacteria, preparation of protoplasts, coating of MOFs artificial cell walls, gradient solvent extraction and product purification.

2. The method according to claim 1, wherein The MOFs material is one of ZIF-8, MIL-101 or UiO-66, and the pore size is 0.5-1.5 nm.

3. The method according to claim 1, characterized in that: The mass ratio of protoplast to MOFs material for assembly is 1-3:1-2.

4. The method according to claim 3, characterized in that: The assembly process conditions of protoplast and MOFs material are: PH = 6.5-7.3, temperature is 28-35 °C, and reaction time is 2-3 hours.

5. The method according to claim 1, characterized in that The gradient solvent extraction includes two-stage extraction of low-polarity solvents (ethyl acetate, n-hexane) and high-polarity solvents (methanol-water, ethanol-water).

6. The method according to claim 5, wherein: The volume ratio of protoplast to low-polarity solvent is: 1:1; the ratio of methanol to water in the high-polarity solvent is: 7:3.

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