Preparation method for synthesizing aminobutyric acid through whole-cell immobilization efficient catalysis

Abstract

The invention relates to the technical field of bioengineering, and provides a preparation method for synthesizing aminobutyric acid through whole-cell immobilization efficient catalysis. The method comprises the following steps: firstly, adding urea as a nitrogen source in the culture process of corynebacterium glutamicum, and feeding glycerol, sorbitol and biotin complex liquid to culture thalli; then carrying out low-temperature incubation treatment on the thalli by using pyridoxal phosphate, and then carrying out mixed incubation with mercapto-histidine trimethyl inner salt; then preparing an embedding carrier solution by using sodium alginate, polyvinyl alcohol and graphene oxide, and adding glutaraldehyde as a cross-linking agent; mixing the treated thalli with an embedding carrier, and adding a mixture of boric acid and calcium chloride; and finally, dropwise adding the mixed solution into a sodium sulfate solution to form gel particles, and storing the gel particles by using an acetic acid-vitamin C composite buffer solution. The immobilized cells prepared by the method have high mechanical strength, high cell encapsulation rate and high conversion rate, are suitable for long-term preservation, still have activity of 90% or more of the original activity after being refrigerated for 10 days, and still have yield of 360 g / L or more after being used for 10 batches.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology. More specifically, the present invention relates to a preparation method for the highly efficient catalytic synthesis of aminobutyric acid by whole-cell immobilization. Background Art

[0002] In recent years, the combination of high-density fermentation and immobilized cell technology has shown significant advantages in the field of bioengineering. This combined technology has many advantages, including improving production efficiency and yield, optimizing the fermentation process and stability, simplifying downstream processing and increasing product purity, as well as expanding application fields and enhancing flexibility.

[0003] By increasing the cell density in the fermenter, the cellular level of the product can be significantly increased, thereby improving the production capacity of the equipment per unit volume. The immobilized cell technology can maintain the original state of the enzyme, improve the enzyme stability, and make the cells more resistant to contamination. In addition, the loss of immobilized cells is less, and continuous fermentation can be carried out at a high dilution rate, improving the equipment utilization rate. This helps to achieve the continuity and automation of the fermentation process and improve production stability. These advantages make this technology combination have broad application prospects and important practical value in the field of microbial fermentation.

[0004] However, during the immobilization process of cells, if only a single immobilization material is used, the obtained immobilized cells usually have low mechanical strength, and the formed gel is prone to autolysis decomposition, softening, floating, and fragmentation. These immobilization materials may contaminate the target product and other problems, ultimately resulting in poor use effects of the immobilized cells.

[0005] L-Histidine, L-threonine betaine (ergothioneine) is a natural amino acid with significant antioxidant properties. It can protect microorganisms from oxidative stress damage, enhance the stress resistance of microorganisms, and promote the growth and metabolism of microorganisms. By scavenging harmful substances such as hydroxyl radicals generated by respiration, it maintains the stability and health of cells. This substance can improve the adaptability of microorganisms to extreme environments such as high temperature and high pressure, and can promote cell proliferation and metabolic activities by stimulating certain enzymes involved in antioxidant reactions and repair mechanisms. In addition, ergothioneine can also serve as a carbon source and nitrogen source for microorganisms, providing necessary nutrients for their growth.

[0006] Despite the above advantages of ergothioneine, how to effectively integrate it into the immobilized cell system and at the same time solve technical problems such as insufficient strength of current immobilization materials to achieve the efficient and stable production of aminobutyric acid is still an urgent challenge to be solved. Summary of the Invention

[0007] To overcome the above-mentioned defects of the prior art, the present invention provides a preparation method for the highly efficient catalytic synthesis of aminobutyric acid by whole-cell immobilization. An alginate, polyvinyl alcohol, graphene oxide composite immobilization material is used, combined with S-methyl-L-histidine inner salt to enhance cell activity, solving the problems of low mechanical strength, easy autolysis decomposition and fragmentation of traditional single immobilization materials. At the same time, it is preserved with a pH 4.6 acetic acid - vitamin C composite buffer, significantly extending the storage time of the catalytic gel particles and improving production efficiency and operational flexibility.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A preparation method for the highly efficient catalytic synthesis of aminobutyric acid by whole-cell immobilization, adding urea, glycerol, sorbitol, and biotin during the preparation process of the highly efficient catalytic synthesis of aminobutyric acid by whole-cell immobilization to culture and prepare a high-density Corynebacterium glutamicum suspension, using S-methyl-L-histidine inner salt and pyridoxal phosphate to incubate and strengthen the cells, and using alginate, polyvinyl alcohol, and graphene oxide to strengthen the immobilization.

[0010] As a further aspect of the present invention, it specifically includes the following steps:

[0011] Step 1, inoculate Corynebacterium glutamicum into a fermentation medium containing urea for fermentation culture, and add a growth adjuvant composed of glycerol, sorbitol, and biotin during the culture process to obtain a fermentation broth;

[0012] Step 2, after centrifugally separating the fermentation broth, remove the supernatant to obtain Corynebacterium glutamicum cells. Add the cells to physiological saline and stir to suspend them. After filtration and washing, sequentially perform low-temperature incubation and strengthening treatment with sterilized low-temperature physiological saline containing pyridoxal phosphate and S-methyl-L-histidine inner salt to obtain a strengthened Corynebacterium glutamicum suspension;

[0013] Step 3, add alginate, polyvinyl alcohol, and graphene oxide to sterile water, heat and dissolve to prepare an embedding carrier solution, and cool it;

[0014] Step 4, add glutaraldehyde to the above-mentioned embedding carrier solution, let it stand, and then add the strengthened Corynebacterium glutamicum suspension prepared in Step 2, and mix evenly to form a mixed solution;

[0015] Step 5, add boric acid and CaCl2 powder to the mixed solution prepared in Step 4, stir and mix to form an immobilized bacterial solution;

[0016] Step 6, drop the immobilized bacterial solution prepared in Step 5 into a Na2SO4 solution in a dropping manner, and after immobilization treatment, form highly efficient catalytic gel particles for whole-cell immobilization;

[0017] Step 7: Wash the whole-cell immobilized highly efficient catalytic gel particles obtained in Step 6 with acetate buffer solution and store them in acetic acid - vitamin C composite buffer solution.

[0018] As a further solution of the present invention, inoculate Corynebacterium glutamicum at 8% into a fermentation medium containing 0.2 - 0.8% urea. The addition of urea can effectively adjust the nitrogen source ratio in the medium.

[0019] The specific formula of the medium includes 60 g / L of glucose and 40 g / L of fructose. These two sugars need to be mixed, sterilized separately, and then added dropwise. It also includes 16 g / L of corn steep liquor powder, 6 g / L of urea, 0.8 g / L of magnesium sulfate, 1 g / L of ferrous sulfate, 1.2 g / L of manganese sulfate, 0.5 g / L of potassium dihydrogen phosphate, 0.1 g / L of dipotassium hydrogen phosphate, 2 g / L of sodium citrate, and the pH value is adjusted to 7.0.

[0020] As a further solution of the present invention, after inoculation, carry out fermentation culture at 37°C and 280 rpm, and at the same time add a growth adjuvant at a rate of 0.6 mL / h. The growth adjuvant is a composite solution of glycerol, sorbitol, and biotin, and its optimal addition ratio is 500000:1500000:1. The specific composition is 5 g / L of glycerol, 15 g / L of sorbitol, and 10 μg / L of biotin. After culturing for 30 - 40 hours, obtain the fermentation broth, and then remove the supernatant by centrifugation to retain the cell precipitate.

[0021] Mix the cell precipitate with 3 - 5 times the mass of sterilized low-temperature physiological saline, stir well to completely suspend the cells, and obtain the washed cells after filtration. This step can effectively remove the residual substances in the medium, provide a pure cell material for subsequent treatment, and thus improve the efficiency of the immobilization process and the product quality.

[0022] As a further solution of the present invention, add the obtained washed cells into sterilized low-temperature physiological saline containing pyridoxal phosphate, mix well, and then carry out low-temperature incubation. This incubation process is carried out at a temperature of 4 - 10°C for 15 - 30 minutes, and the optimal incubation conditions are 6°C and 20 minutes. The addition amount of pyridoxal phosphate is 0.1 - 0.7% of the mass of the cell suspension, and its optimal addition amount is 0.3%. This step can significantly enhance the activity and stability of Corynebacterium glutamicum.

[0023] As a further embodiment of the present invention, after the incubation is completed, ergothioneine is added to the bacterial suspension, and the addition amount is 0.2-1.2%, and the optimal addition amount is 0.8%. After mixing, continue to incubate at 30°C for 20-60 minutes, and the optimal incubation time is 35 minutes. As a natural amino acid with significant antioxidant properties, ergothioneine can protect microorganisms from oxidative stress damage, enhance the stress resistance of microorganisms, and at the same time promote their growth and metabolism. By scavenging harmful substances such as hydroxyl radicals generated by respiration, it maintains the stability and health of cells.

[0024] As a further embodiment of the present invention, in the process of preparing whole-cell immobilized high-efficiency catalytic synthesis of γ-aminobutyric acid, the preparation of the immobilization material is a key step, mainly by preparing an embedding carrier solution to prepare for subsequent cell immobilization. Specifically, sodium alginate, polyvinyl alcohol and graphene oxide are mixed in a mass ratio of 15:200:18, and their optimal addition amounts are 1.5%, 3% and 1.8% respectively. This composite material with a specific ratio can significantly enhance the immobilization effect.

[0025] In actual operation, weigh 0.3 g of sodium alginate, 4 g of polyvinyl alcohol, and 0.36 g of graphene oxide and add them to 100 mL of sterile water. Heat and dissolve at a temperature of 60-80°C, and fully mix them to completely dissolve to form a uniform solution. Subsequently, cool the solution to 40-50°C and keep it warm to obtain the embedding carrier solution. This temperature control process is crucial for maintaining the appropriate viscosity and activity of the embedding carrier solution, which is beneficial to the subsequent full mixing and embedding with cells.

[0026] This composite embedding carrier solution containing sodium alginate, polyvinyl alcohol and graphene oxide breaks through the limitations of traditional single immobilization materials, and effectively solves problems such as low mechanical strength, easy autolysis decomposition, softening, floating, and fragmentation of immobilized cells. Especially the addition of graphene oxide enhances the strength and stability of the entire immobilization material, laying a foundation for the subsequent preparation of high-quality whole-cell immobilized high-efficiency catalytic gel particles.

[0027] As a further embodiment of the present invention, after obtaining the embedding carrier solution, crosslinking and cell mixing treatment are required. The specific operation is to add 1.5 mL of glutaraldehyde as a crosslinking agent to the previously prepared embedding carrier solution, and let it stand at a temperature of 25-35°C for 30-50 minutes to allow glutaraldehyde to fully crosslink with the active groups in the embedding carrier solution. This standing process can enable the embedding carrier solution to form a preliminary network structure, providing a better matrix environment for subsequent cell embedding.

[0028] After standing still, add the aforementioned Corynebacterium glutamicum suspension that has been intensively treated, and place the mixture in a water bath shaker incubator for thorough mixing. The mixing conditions are shaking at 30°C and 130 - 160 rpm for 80 - 100 minutes. During this process, the volume ratio of the sodium alginate solution to the Corynebacterium glutamicum suspension is maintained at 1:1, and the OD600 value of the used Corynebacterium glutamicum suspension should be within the range of 200 - 300. Through mixing under these specific conditions, the intensively treated cells can be evenly dispersed in the embedding carrier solution, forming a stable mixing system, which lays the foundation for subsequent immobilization treatment.

[0029] As a further scheme of the present invention, after completing the oscillating mixing, it will enter the preparation stage of the immobilized bacterial liquid. At this time, add a mixture of boric acid and CaCl2 powder with a specific ratio to the mixed liquid. The mass ratio of boric acid to CaCl2 is 4:1, and the mass fraction of boric acid is controlled within the range of 3.2 - 4.4%, and the optimal addition amount is 3.6%. In actual operation, usually 3.6 g of boric acid and 0.9 g of CaCl2 powder are mixed and then added to the aforementioned mixed liquid, and stirred thoroughly for 10 - 20 minutes to completely dissolve and disperse evenly.

[0030] Boric acid and CaCl2 undergo an ion cross-linking reaction with components such as sodium alginate in the embedding carrier solution, further enhancing the mechanical strength and stability of the immobilized material. Especially the addition of boric acid can form a complex borate cross-linking structure with polyvinyl alcohol, improving the hardness and durability of the immobilized particles.

[0031] As a further scheme of the present invention, after obtaining the immobilized bacterial liquid, it is necessary to carry out dropping immobilization treatment to form whole-cell immobilized highly efficient catalytic gel particles. The specific operation is to suck the immobilized bacterial liquid into a sterilized syringe and inject it dropwise into a 16% concentration Na2SO4 solution at a height of 8 cm from the liquid surface for immobilization. During the dropping process, the volume ratio of the immobilized bacterial liquid to the Na2SO4 solution should be controlled at 1:8 - 10, and this ratio can ensure sufficient ion exchange and gel formation.

[0032] In the Na2SO4 solution, Ca 2+ ions in the immobilized bacterial liquid react with SO4 2- ions, and at the same time, Na + ions act with alginate ions to form gel particles with a three-dimensional network structure. During this process, the cross-linking network formed by boric acid and polyvinyl alcohol interweaves with the ion cross-linking network, further enhancing the mechanical strength and stability of the gel particles. After the immobilization process is completed, whole-cell immobilized highly efficient catalytic gel particles with high strength and high stability are obtained, in which Corynebacterium glutamicum cells are evenly embedded inside the gel particles.

[0033] As a further solution of the present invention, after forming the whole-cell immobilized highly efficient catalytic gel particles, cleaning and preservation treatments are required to ensure their long-term activity and stability. First, the gel particles are thoroughly cleaned with an acetic acid buffer solution with a pH of 4.6 to remove substances such as residual Na2SO4 on the particle surface. The specific formulation of this acetic acid buffer solution is 18 g of sodium acetate, 9.8 mL of glacial acetic acid, and made up to 1 L with ultrapure water. The cleaning solution with this specific pH value can effectively remove surface impurities without having an adverse effect on the activity of the immobilized cells.

[0034] After cleaning, the gel particles are transferred to an acetic acid-VC composite buffer solution with a pH of 4.6 and stored at 4°C. The formulation of this composite buffer solution includes 0.5 g of vitamin C, 11.6 g of K2HPO4, 0.88 g of sodium citrate, 0.16 g of MgSO4, 3.4 g of KH2PO4, and 88 g of glycerol, and made up to 1 L with an acetic acid buffer solution with a pH of 4.6. This preservation solution with a special formulation has a significant protective effect. In particular, the addition of vitamin C can effectively scavenge oxygen free radicals in the environment and protect the immobilized cells from oxidative damage. The experimental results show that the activity of the immobilized cells stored in the acetic acid-VC composite buffer solution is relatively stable, and more than 90% of the activity can still be maintained after 10 days of refrigeration, while the activity of the cells stored only in the acetic acid buffer solution drops below 90% on the 4th day and below 80% on the 7th day.

[0035] Compared with the prior art, the beneficial effects of the preparation method of the present invention for the whole-cell immobilized highly efficient catalytic synthesis of γ-aminobutyric acid are as follows:

[0036] The present invention adopts a composite immobilization material system of sodium alginate, polyvinyl alcohol, and graphene oxide, and adds S-methyl-L-histidine to enhance cell activity. Compared with the application of a single immobilization material in the prior art, this composite system significantly enhances the mechanical strength of the immobilized gel particles, effectively avoids problems such as gel autolysis decomposition, softening, floating, and fragmentation, improves the encapsulation rate of the immobilized cells, and enhances the catalytic efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a process schematic diagram of the preparation method of the present invention for the whole-cell immobilized highly efficient catalytic synthesis of γ-aminobutyric acid.

[0038] Figure 2 It is a high-performance liquid chromatography analysis chart of γ-aminobutyric acid of the present invention.

[0039] Figure 3 It is a comparison chart of the repeated use yields of the immobilized cells and free cells of the present invention.

[0040] Figure 4 It is a diagram showing the effect of different preservation buffer solutions of the present invention on the activity of immobilized cells.

[0041] Figure 5 This is a comparison graph of the storage stability between the immobilized cells and free cells of the present invention. Detailed implementation manners

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0043] Example 1

[0044] In the embodiment of the present invention, first, Corynebacterium glutamicum was inoculated into a fermentation medium supplemented with 0.6% urea at 8%. The composition of the used medium was prepared by mixing 60 g / L of glucose and 40 g / L of fructose, sterilized separately and then fed in, and at the same time, 16 g / L of corn steep powder, 6 g / L of urea, 0.8 g / L of magnesium sulfate, 1 g / L of ferrous sulfate, 1.2 g / L of manganese sulfate, 0.5 g / L of potassium dihydrogen phosphate, 0.1 g / L of dipotassium hydrogen phosphate, and 2 g / L of sodium citrate were added, and the pH value was adjusted to 7.0. Subsequently, the medium was fermented and cultured at 37°C and 280 rpm, and a growth adjuvant was fed at a rate of 0.6 mL / h during the culture process. The growth adjuvant was a composite solution of 5 g / L of glycerol, 15 g / L of sorbitol, and 10 μg / L of biotin. After 36 hours of culture, a fermented bacterial liquid was obtained.

[0045] The obtained fermented bacterial liquid was centrifuged to remove the supernatant, and the cell precipitate part was retained. Then, 4 times the mass of the cells of sterilized low-temperature physiological saline was added to the obtained cells, and they were fully stirred to completely suspend the cells. After filtration, the washed pure cells were obtained. Subsequently, the washed cells were added to sterilized low-temperature physiological saline containing pyridoxal phosphate, and the addition amount of pyridoxal phosphate was 0.3% of the mass of the cell suspension. After thorough mixing, it was incubated at 6°C for 20 minutes. This process can significantly enhance the activity and stability of Corynebacterium glutamicum. After the incubation was completed, S-methyl-L-cysteine was added to the cell suspension, and the addition amount was 0.8% of the cell suspension. After mixing again, it was incubated at 30°C for 50 minutes to obtain a cell suspension.

[0046] Next, the embedding carrier solution was prepared. 0.3g of sodium alginate, 4g of polyvinyl alcohol and 0.36g of graphene oxide were weighed, and the three materials were added to 100mL of sterile water, fully heated and dissolved at 65°C to prepare a uniform embedding carrier solution, which was then cooled to 45°C for insulation. After the embedding carrier solution was cooled, 1.5mL of glutaraldehyde was added thereto as a cross-linking agent, and the solution was allowed to stand for 36 minutes to allow glutaraldehyde to fully cross-link with the active groups in the embedding carrier solution. After standing, the aforementioned prepared fortified bacterial suspension was added, and the whole mixture was placed under 30°C and 150rpm conditions for oscillation mixing, and the time was controlled at 80 minutes to ensure that the fortified cells were evenly dispersed in the embedding carrier solution, and a stable and uniform mixed solution was finally obtained.

[0047] After the mixing is completed, 3.6g of boric acid and 0.9g of CaCl2 powder are added to the mixed solution, and the mixture is stirred for 15 minutes to fully dissolve and evenly disperse to obtain an immobilized bacterial solution. The addition of boric acid and CaCl2 can react with the sodium alginate and other components in the embedding carrier solution to produce an ionic crosslinking reaction, which significantly enhances the mechanical strength and stability of the immobilized material. The prepared immobilized bacterial solution is then sucked into a sterilized syringe, and a 16% concentration of Na2SO4 solution is injected dropwise at a height of 8cm from the liquid surface for immobilization, while the volume ratio of the immobilized bacterial solution to the Na2SO4 solution is controlled within the range of 1:8-10. Through this treatment, the immobilized bacterial solution forms gel particles of uniform size and shape in the Na2SO4 solution, wherein the Corynebacterium glutamicum cells are firmly embedded inside the gel particles.

[0048] Finally, the formed gel particles were thoroughly washed with an acetate buffer solution at pH 4.6, which was prepared from 18 g of sodium acetate, 9.8 mL of glacial acetic acid, and ultrapure water fixed to 1 L. After washing, the gel particles were transferred to an acetate-vitamin C complex buffer solution at pH 4.6 and stored at 4°C. This special preservation solution consists of 0.5 g vitamin C, 11.6 g K2HPO4, 0.88 g sodium citrate, 0.16 g MgSO4, 3.4 g KH2PO4, and 88 g glycerol, and an acetate buffer solution at pH 4.6 was added to fix the volume to 1 L.

[0049] Example 2

[0050] The method for preparing glutamate-immobilized cells comprises the following steps:

[0051] Step 1: Inoculate Corynebacterium glutamicum at 8% into a fermentation medium supplemented with 0.6% urea, ferment and culture at 37°C and 280 rpm, feed a compound solution of glycerol, sorbitol and biotin at 0.6 mL / h and culture for 36 h to obtain a fermentation broth. Centrifuge the fermentation broth, discard the supernatant, retain the cells, add 4 times the mass of sterilized low-temperature physiological saline to the cells, stir to fully suspend the cells, filter to obtain the washed cells.

[0052] Step 2: Add the washed cells into sterilized low-temperature physiological saline containing pyridoxal phosphate, mix well and incubate at low temperature for 20 min, add S-methyl-L-histidine inner salt and mix well, incubate at 30°C for 50 min to prepare a cell suspension.

[0053] Step 3: Weigh 0.3 g of sodium alginate, 4 g of polyvinyl alcohol, and 0.36 g of graphene oxide, add them to 100 mL of sterile water at 65°C, heat and dissolve to prepare an embedding carrier solution, cool to 45°C and keep warm to obtain the embedding carrier solution.

[0054] Step 4: Add 1.5 mL of glutaraldehyde to the embedding carrier solution and let it stand for 36 min, add the incubated cell suspension, place it in a water bath shaking incubator and mix well, shake at 30°C and 150 rpm for 80 minutes to mix evenly to obtain a mixed solution.

[0055] Step 5: Add 3.6 g of boric acid and 0.9 g of CaCl2 powder to the mixed solution, stir the mixed solution for 15 min to obtain an immobilized cell solution.

[0056] Step 6: Drop the immobilized cell solution into a 16% Na2SO4 solution for immobilization to obtain whole-cell immobilized highly efficient catalytic gel particles.

[0057] In the embodiment of the present invention, the prepared whole-cell immobilized highly efficient catalytic gel particles are used for the conversion experiment of glutamic acid to γ-aminobutyric acid. First, weigh 1290 g of glutamic acid with a purity of 98%, add 1 L of water, stir evenly and put it into a 5 L fermentation tank. Then add the highly efficient catalytic gel particles of Corynebacterium glutamicum prepared according to the method of Example 1 into the fermentation tank, and stir and culture at 37°C and 300 rpm.

[0058] After 48 hours of continuous culture, the whole conversion reaction is ended. After the reaction is completed, separate the highly efficient catalytic gel particles of Corynebacterium glutamicum from the fermentation broth to obtain a fermentation broth containing γ-aminobutyric acid. As Figure 2 shown, the whole-cell immobilized highly efficient catalytic gel particles prepared by the present invention can effectively convert glutamic acid into γ-aminobutyric acid and have good catalytic effects.

[0059] Example 3

[0060] A method for preparing glutamic acid-immobilized cells, comprising the following steps:

[0061] Step 1: Inoculate Corynebacterium glutamicum at 8% into a fermentation medium supplemented with 0.6% urea, ferment and culture at 37°C and 280 rpm. Feed a compound solution of glycerol, sorbitol and biotin at 0.6 mL / h and culture for 36 h to obtain a fermentation broth. Centrifuge the fermentation broth, discard the supernatant, retain the cells, add 4 times the mass of sterilized low-temperature physiological saline to the cells, stir to fully suspend the cells, and filter to obtain the washed cells.

[0062] Step 2: Add the washed cells to sterilized low-temperature physiological saline containing pyridoxal phosphate, mix well and incubate at low temperature for 20 min. Add S-methyl-L-histidine and mix well, then incubate at 30°C for 50 min to prepare a cell suspension.

[0063] Step 3: Weigh 0.3 g of sodium alginate, 4 g of polyvinyl alcohol and 0.36 g of graphene oxide, add them to 100 mL of sterile water at 65°C, heat and dissolve to prepare an embedding carrier solution, cool to 45°C and keep warm to obtain the embedding carrier solution.

[0064] Step 4: Add 1.5 mL of glutaraldehyde to the embedding carrier solution and let it stand for 36 min. Add the incubated cell suspension and place it in a water bath shaker incubator to mix well. Shake at 30°C and 150 rpm for 80 minutes to mix evenly and obtain a mixed solution.

[0065] Step 5: Add 3.6 g of boric acid and 0.9 g of CaCl2 powder to the mixed solution, and stir the mixed solution for 15 min to obtain an immobilized cell solution.

[0066] Step 6: Drop the immobilized cell solution into a 16% Na2SO4 solution for immobilization to obtain whole-cell immobilized highly efficient catalytic gel particles.

[0067] To explore the industrial application potential of immobilized cells, experiments were carried out using immobilized cells and free cells to convert glutamic acid to prepare γ-aminobutyric acid. As Figure 3 shown, when the free cells were used in the fourth batch, the yield was only 364 g / L. After six batches, the catalytic ability was basically lost. While the yield of the immobilized cells remained above 403 g / L in the first 6 conversion batches. When reused to the 10th batch, the yield was 366 g / L. Compared with free cells, the operation stability and catalytic efficiency of immobilized cells were significantly improved.

[0068] Example 4

[0069] A method for preparing immobilized glutamic acid cells, comprising the following steps:

[0070] Step 1: Inoculate Corynebacterium glutamicum at 8% into a fermentation medium supplemented with 0.6% urea, ferment and culture at 37°C and 280 rpm, feed a compound solution of glycerol, sorbitol and biotin at 0.6 mL / h and culture for 36 h to obtain a fermentation broth. Centrifuge the fermentation broth, discard the supernatant, retain the bacterial cells, add 4 times the mass of sterilized low-temperature physiological saline to the bacterial cells, stir to fully suspend the bacterial cells, and filter to obtain the washed bacterial cells;

[0071] Step 2: Add the washed bacterial cells into sterilized low-temperature physiological saline containing pyridoxal phosphate, mix well and incubate at low temperature for 20 min, add S-methyl-L-histidine and mix well, then incubate at 30°C for 50 min to prepare a bacterial suspension;

[0072] Step 3: Weigh 0.3 g of sodium alginate, 4 g of polyvinyl alcohol, and 0.36 g of graphene oxide, add them to 100 mL of sterile water at 65°C, heat and dissolve to prepare an embedding carrier solution, cool to 45°C and keep warm to obtain the embedding carrier solution;

[0073] Step 4: Add 1.5 mL of glutaraldehyde to the embedding carrier solution and let it stand for 36 min, add the incubated bacterial suspension, place it in a water bath shaking incubator and mix well, shake at 30°C and 150 rpm for 80 minutes to mix evenly and obtain a mixed solution;

[0074] Step 5: Add 3.6 g of boric acid and 0.9 g of CaCl2 powder to the mixed solution, stir the mixed solution for 15 min to obtain an immobilized bacterial solution.

[0075] Step 6: Drop the immobilized bacterial solution into a 16% Na2SO4 solution for immobilization to obtain whole-cell immobilized highly efficient catalytic gel particles.

[0076] In the examples of the present invention, to explore the influence of different preservation conditions on the activity of immobilized cells, the whole-cell immobilized highly efficient catalytic gel particles prepared in the same batch were preserved at 4°C using an acetic acid - vitamin C composite buffer solution with pH 4.6 and an acetic acid solution with pH 4.6, as Figure 4 shown. After detection, the activity of the immobilized cells preserved in the acetic acid - vitamin C composite buffer solution was relatively stable, all maintained above 90%. The activity of the immobilized cells preserved in the acetic acid solution was lower than 90% on the 4th day and lower than 80% on the 7th day.

[0077] Example 5

[0078] A method for preparing glutamic acid immobilized cells, comprising the following steps:

[0079] Step 1: Inoculate Corynebacterium glutamicum at 8% into a fermentation medium supplemented with 0.6% urea, ferment and culture at 37°C and 280 rpm. Feed a complex solution of glycerol, sorbitol, and biotin at 0.6 mL / h and culture for 36 h to obtain a fermentation broth. Centrifuge the fermentation broth, discard the supernatant, retain the cells, add 4 times the mass of sterilized low-temperature physiological saline to the cells, stir to fully suspend the cells, and filter to obtain the washed cells.

[0080] Step 2: Add the washed cells to sterilized low-temperature physiological saline containing pyridoxal phosphate, mix well and incubate at low temperature for 20 min. Add S-methyl-L-cysteine and mix well, then incubate at 30°C for 50 min to prepare a cell suspension.

[0081] Step 3: Weigh 0.3 g of sodium alginate, 4 g of polyvinyl alcohol, and 0.36 g of graphene oxide, add them to 100 mL of sterile water at 65°C, heat and dissolve to prepare an embedding carrier solution, cool to 45°C and keep warm to obtain the embedding carrier solution.

[0082] Step 4: Add 1.5 mL of glutaraldehyde to the embedding carrier solution and let it stand for 36 min. Add the incubated cell suspension, place it in a water bath shaker incubator and mix well. Shake at 30°C and 150 rpm for 80 minutes to mix evenly and obtain a mixed solution.

[0083] Step 5: Add 3.6 g of boric acid and 0.9 g of CaCl2 powder to the mixed solution, and stir the mixed solution for 15 min to obtain an immobilized cell solution.

[0084] Step 6: Drop the immobilized cell solution into a 16% Na2SO4 solution for immobilization to obtain whole-cell immobilized highly efficient catalytic gel particles.

[0085] In the examples of the present invention, in order to further evaluate the effect of the immobilization treatment on the long-term preservation performance of cells, the storage stability of the immobilized cells was studied; this is an important indicator for its industrial application. In the experiment, the immobilized cells prepared according to the above steps and the corresponding free cells were simultaneously stored at 4°C, and the changes in the activities of the two types of cells within 14 days were observed. As Figure 5 shown, the activity of the immobilized cells could still be maintained at about 90% in the first 10 days of storage, while the activity of the free cells under the corresponding conditions had decreased to less than 30%. When the storage period was extended to 14 days, the immobilized cells could still maintain 70% of their activity, while the remaining activity of the free cells was hardly detectable. This result fully demonstrates the significant effect of the immobilization method of the present invention on improving the long-term storage stability of cells, providing the possibility for its industrial production application.

[0086] As described above, it is only the specific implementation manner of the present application. However, the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present application can easily think of changes or substitutions, which should all be covered within the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claimed rights.

[0087] Finally: The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method for highly efficient catalytic synthesis of gamma-aminobutyric acid by whole-cell immobilization, characterized in that, During the preparation of whole-cell immobilized high-efficiency catalytic synthesis of γ-aminobutyric acid, urea, glycerol, sorbitol, and biotin were added to culture and prepare a high-density suspension of Corynebacterium glutamicum. Thiolhistidine trimethyl inner salt and pyridoxal phosphate were used to incubate and strengthen the cells, and sodium alginate, polyvinyl alcohol, and graphene oxide were used to strengthen the immobilization.

2. The preparation method for highly efficiently catalytically synthesizing aminobutyric acid by whole-cell immobilization according to claim 1, characterized in that, Specifically, it includes the following steps: Step 1, inoculate Corynebacterium glutamicum into a fermentation medium containing urea for fermentation culture, and add a growth adjuvant composed of glycerol, sorbitol, and biotin during the culture process to obtain a fermentation broth; Step 2, after centrifugally separating the fermentation broth, remove the supernatant to obtain Corynebacterium glutamicum cells. Add the cells to physiological saline and stir to suspend them. After filtration and washing, successively perform low-temperature incubation and strengthening treatment with sterilized low-temperature physiological saline containing pyridoxal phosphate and thiolhistidine trimethyl inner salt to prepare a strengthened suspension of Corynebacterium glutamicum; Step 3, add sodium alginate, polyvinyl alcohol, and graphene oxide to sterile water, heat and dissolve to prepare an embedding carrier solution, and cool it; Step 4, add glutaraldehyde to the embedding carrier solution, let it stand, and then add the strengthened suspension of Corynebacterium glutamicum prepared in Step 2, and mix evenly to form a mixed solution; Step 5, add boric acid and CaCl2 powder to the mixed solution prepared in Step 4, and stir and mix to form an immobilized bacterial solution; Step 6, drop the immobilized bacterial solution prepared in Step 5 into a Na2SO4 solution in a dropping manner, and form whole-cell immobilized high-efficiency catalytic gel particles after immobilization treatment; Step 7, wash the whole-cell immobilized high-efficiency catalytic gel particles obtained in Step 6 with acetic acid buffer solution, and store them in an acetic acid-VC composite buffer solution.

3. The preparation method for highly efficiently catalytically synthesizing aminobutyric acid by whole-cell immobilization according to claim 2, wherein In Step 1, the composition of the fermentation medium is: mix 60 g / L of glucose and 40 g / L of fructose, sterilize them separately and then add them dropwise. At the same time, the medium also contains 16 g / L of corn steep powder, 6 g / L of urea, 0.8 g / L of magnesium sulfate, 1 g / L of ferrous sulfate, 1.2 g / L of manganese sulfate, 0.5 g / L of potassium dihydrogen phosphate, 0.1 g / L of dipotassium hydrogen phosphate, and 2 g / L of sodium citrate, and adjust the pH value to 7.

0.

4. The preparation method for highly efficiently catalytically synthesizing aminobutyric acid by whole-cell immobilization according to claim 2, wherein, The Corynebacterium glutamicum is inoculated into a fermentation medium containing 0.2 - 0.8% urea at an inoculation amount of 8%; the conditions for fermentation culture are 37°C, 280 rpm, and the culture time is 30 - 40 h; the ratio of glycerol, sorbitol, and biotin in the growth adjuvant is 500000:1500000:1, and the flow rate is 0.6 mL / h.

5. A preparation method for highly efficient catalytic synthesis of aminobutyric acid by whole-cell immobilization according to claim 2, characterized in that, In Step 2, the cells are added to 3 - 5 times of sterilized low-temperature physiological saline and stirred to suspend them; after washing, the Corynebacterium glutamicum cells are mixed with sterilized physiological saline containing pyridoxal phosphate and incubated at 4 - 10°C for 15 - 30 min. The addition amount of pyridoxal phosphate is 0.1 - 0.7% of the mass of the bacterial suspension; the addition amount of thiolhistidine trimethyl inner salt is 0.2 - 1.2%; the incubation conditions of the Corynebacterium glutamicum cells and thiolhistidine trimethyl inner salt are 30°C for 20 - 60 minutes.

6. The preparation method for highly efficiently catalytically synthesizing aminobutyric acid by whole-cell immobilization according to claim 2, characterized in that, In step three, the mass ratio of sodium alginate, polyvinyl alcohol, and graphene oxide is 15:200:

18. After heating and dissolving in sterile water at 60 - 80 °C and then cooling, an embedding carrier solution is prepared and kept warm at 40 - 50 °C. The addition amounts of sodium alginate, polyvinyl alcohol, and graphene oxide are 1.5%, 3%, and 1.8% respectively.

7. The preparation method for highly efficiently catalytically synthesizing aminobutyric acid by whole-cell immobilization according to claim 2, characterized in that, In step four, 1.5 mL of glutaraldehyde is added to the embedding carrier solution and left standing at 25 - 35 °C for 30 - 50 minutes. The mixing conditions after adding the Corynebacterium glutamicum suspension are 30 °C and shaking and mixing at 130 - 160 rpm for 80 - 100 minutes. The volume ratio of the sodium alginate solution to the Corynebacterium glutamicum suspension is 1:1, and the OD600 of the Corynebacterium glutamicum suspension is 200 - 300.

8. The preparation method for highly efficiently catalytically synthesizing aminobutyric acid by whole-cell immobilization according to claim 2, wherein, In step five, the mass ratio of boric acid to CaCl2 is 4:1, and the mass fraction of boric acid is 3.2 - 4.4%. After adding the boric acid and CaCl2 powder mixture to the shaken and mixed solution, it is stirred for 10 - 20 minutes to obtain the immobilized bacterial solution.

9. The preparation method for highly efficiently catalytically synthesizing aminobutyric acid by whole-cell immobilization according to claim 2, characterized in that, In step six, the concentration of the Na2SO4 solution is 16%. The dropping method of the immobilized bacterial solution is to suck the immobilized bacterial solution into a sterilized syringe and drop it into the 16% Na2SO4 solution for immobilization at a distance of 8 cm from the liquid surface. And the volume ratio of the dropped immobilized bacterial solution to the 16% Na2SO4 solution is 1:8 - 10.

10. The preparation method for highly efficient catalytic synthesis of aminobutyric acid by whole-cell immobilization according to claim 2, characterized in that, In step seven, the pH of the acetate buffer solution used to wash the whole-cell immobilized highly efficient catalytic gel particles is 4.

6. The obtained whole-cell immobilized highly efficient catalytic gel particles are stored in an acetic acid - vitamin C composite buffer solution with a pH of 4.6 at 4 °C.

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