A high-stability allulose-3-epimer fermentation preparation method

Abstract

The present application belongs to the technical field of enzyme preparation, and particularly relates to a high-stability allulose-3-epimerase fermentation preparation method. In the logarithmic growth phase of the recombinant bacteria fermentation, arginine and glutamic acid composition is supplemented to the basic medium, and a manganese ion-containing ethylenediamine bis-o-hydroxyphenylacetic acid complex is coupled and added. The above composition is added in the form of gelatin and sodium alginate cross-linked polymer slow-release microcapsules, and the complex is added in stages with interval. After the supplement, the stirring speed and the air-pure oxygen mixed gas composition are jointly adjusted to maintain a specific dissolved oxygen level to the fermentation endpoint. Through the amino acid intervention of the new peptide chain folding microenvironment, the generation of inclusion bodies is inhibited, and the chelated manganese ion acts as a cofactor precursor to bind to the enzyme protein active center in situ in the cell, thereby improving the soluble enzyme expression proportion and the cofactor loading rate. The problem of insufficient enzyme preparation thermal stability caused by protein misfolding and low intracellular cofactor loading rate in the conventional fermentation process is solved.

Description

Technical Field

[0001] This invention belongs to the field of enzyme preparation technology, specifically relating to a fermentation preparation method for a highly stable allulose-3-epimerase. Background Technology

[0002] Currently, the industrial preparation of allulose epimerase mainly utilizes high-density fermentation with genetically engineered bacteria. In conventional fermentation processes, the expression of the exogenous gene is initiated by adding an inducer when the cells reach the late logarithmic growth phase. At this stage, free divalent manganese ions are directly added to the fermentation system as a cofactor for the enzyme. Simultaneously, to maintain high-density cell growth and expression, the dissolved oxygen level in the fermentation broth is typically maintained by increasing the stirring speed or aeration rate. After fermentation, the fermentation broth is directly centrifuged to obtain the supernatant or mechanically crushed to obtain the crude enzyme solution.

[0003] In the aforementioned conventional fermentation process, the rapid expression of exogenous genes during the induction phase leads to the folding speed of newly formed peptide chains in recombinant bacteria not keeping up with the translation speed. Some enzyme proteins that have not completed proper folding aggregate due to hydrophobic interactions, forming inclusion body precipitates. At the same time, the directly added free divalent manganese ions in the fermentation broth are prone to precipitation reactions with components such as phosphates, resulting in a limited number of manganese ions entering the cell and binding to the active site of the enzyme protein. Consequently, the allulose epimerase obtained in the end has the core defect of insufficient thermal stability. Summary of the Invention

[0004] To address the shortcomings of existing allulose-3-epimerase fermentation preparation processes, such as the tendency for nascent peptide chains to fold incorrectly, leading to inclusion bodies, and the easy precipitation of free divalent manganese ions resulting in low cofactor loading rates and insufficient thermal stability of the final enzyme, this invention provides a highly stable allulose-3-epimerase fermentation preparation method.

[0005] To address the aforementioned technical problems, this invention provides a method for preparing highly stable allulose-3-epimerase by fermentation. The method includes the following steps: inoculating a recombinant bacterium containing an allulose-3-epimerase gene into a basal culture medium for fermentation; during the late logarithmic growth phase of the recombinant bacterium's fermentation, supplementing the basal culture medium with a combination of L-arginine and L-glutamic acid, and coupling the addition of a chelated preparation containing divalent manganese ions; the final mass ratio of L-arginine to L-glutamic acid in the basal culture medium is 1:(1.5-3.0), and the final concentration of the chelated preparation containing divalent manganese ions in the basal culture medium is 0.5-2.0 mmol / L; after the addition of the combination and the chelated preparation, adjusting and maintaining the dissolved oxygen level of the fermentation system at 20%-30% until the fermentation endpoint, and collecting the fermentation broth.

[0006] L-arginine and L-glutamic acid, as amino acids with molecular chaperone activity, can enter the folding microenvironment of nascent peptide chains within recombinant bacterial cells. Through electrostatic interactions, they bind to the hydrophobic regions on the surface of unfolded peptide chains, inhibiting hydrophobic aggregation between peptide chains, reducing inclusion body formation, and significantly improving the correct folding rate of allulose-3-epimerase protein. Chelated divalent manganese ions can avoid precipitation reactions with anions such as phosphate and carbonate in the fermentation medium, and can cross the membrane into the recombinant bacterial cell as intact chelate molecules. Under intracellular physiological conditions, they hydrolyze to release free divalent manganese ions, which bind in situ to the active site of allulose-3-epimerase, completing the efficient loading of metal cofactors and stabilizing the spatial conformation of the enzyme protein. A dissolved oxygen level of 20%-30% can maintain the energy supply required for basal metabolism and protein synthesis of the bacteria while avoiding the accumulation of intracellular reactive oxygen species caused by high dissolved oxygen, reducing oxidative damage to amino acid residues of nascent peptide chains by reactive oxygen species, and providing a suitable intracellular microenvironment for the correct folding of the enzyme protein and the stable embedding of cofactors.

[0007] Furthermore, in the above technical solution, the late stage of the logarithmic growth phase is determined by online monitoring of the biomass and pH value of the fermentation broth. When the optical density value corresponding to the biomass of the fermentation broth reaches 8-10 times the optical density value at the initial inoculation, and the pH value of the fermentation broth shows an inflection point in the rate of decrease, the addition of the composition and the chelated preparation is performed.

[0008] In practice, the timing of this supplementation corresponds to the recombinant bacterial cell concentration reaching a high-density threshold and being at the critical point between the end of logarithmic growth and the induction of exogenous protein expression. At this time, the activity of the bacterial protein translation system reaches its peak. Early intervention in the peptide chain folding environment and cofactor supply can precisely match the induction process of exogenous genes, avoiding the problems of inhibiting bacterial growth due to premature intervention or being unable to reverse peptide chain misfolding due to late intervention. At the same time, the inflection point of the pH decrease rate can accurately reflect the physiological state switch of the bacteria from carbon source decomposition metabolism to protein synthesis metabolism, ensuring that the supplementation operation is highly adapted to the physiological state of the bacteria.

[0009] Furthermore, in the above technical solution, the composition of L-arginine and L-glutamic acid is added in the form of sustained-release microcapsules. The capsule wall material of the sustained-release microcapsules is a cross-linked polymer of gelatin and sodium alginate. The particle size of the sustained-release microcapsules is 50-100 micrometers, and the release half-life of the sustained-release microcapsules in the basal culture medium is 2-4 hours.

[0010] In practice, the cross-linked polymer of gelatin and sodium alginate has good biocompatibility and pH responsiveness to the fermentation system. It can be slowly and controllably degraded in the neutral pH environment of the fermentation system to achieve uniform release of the amino acid composition. The particle size of 50-100 micrometers and the release half-life of 2-4 hours can accurately match the complete cycle of exogenous protein expression in recombinant bacteria, avoid the sudden increase in local osmotic pressure in the fermentation system caused by a one-time addition of amino acids, reduce the physiological impact on the bacteria, and at the same time continuously maintain the concentration of amino acid microenvironment required for intracellular peptide chain folding, ensuring the sustainability of the folding intervention effect.

[0011] Furthermore, in the above technical solution, the sustained-release microcapsule also contains L-proline, the mass ratio of L-proline to L-arginine is 1:2, the final concentration of L-proline added to the basal culture medium is 0.1-0.5 g / L, and the sustained-release microcapsule is pumped into the basal culture medium at a constant flow rate using an injection pump.

[0012] In practice, L-proline, as a compatible solute, can work synergistically with L-arginine and L-glutamic acid to stabilize the folding intermediate of the peptide chain through hydrogen bonding, thereby further reducing the probability of peptide chain misfolding and aggregation. The constant flow rate of the injection pump can precisely control the replenishment rate of the microcapsules, avoiding drastic fluctuations in the amino acid concentration within the system, while further reducing the negative impact of local osmotic pressure shocks on bacterial growth and exogenous protein expression.

[0013] Furthermore, in the above technical solution, the chelated preparation containing divalent manganese ions is an aqueous solution of a complex of divalent manganese ions and ethylenediamine di-o-hydroxyphenylacetic acid, wherein the molar ratio of divalent manganese ions to ethylenediamine di-o-hydroxyphenylacetic acid in the aqueous solution of the complex is 1:1.2, and the aqueous solution of the complex is subjected to sterilization filtration through a 0.22-micron filter membrane before being added.

[0014] In practice, ethylenediamine di-o-hydroxyphenylacetic acid has a high selective chelating ability for divalent manganese ions. The molar ratio of 1:1.2 ensures that divalent manganese ions are completely chelated, avoiding the presence of free manganese ions. This blocks the precipitation reaction of manganese ions with anions such as phosphate and carbonate in the fermentation medium, ensuring the stability and bioavailability of manganese ions in the fermentation system. The 0.22-micron filter membrane sterilization filtration can thoroughly remove exogenous bacteria from the complex aqueous solution, preventing product failure and fermentation failure caused by bacterial contamination during fermentation.

[0015] Furthermore, in the above technical solution, the addition of the composition and the chelated preparation adopts a staged addition strategy. The composition is added in the first stage for 1 hour, and the chelated preparation is added in the second stage for 0.5 hours. There is a 30-minute interval between the first stage and the second stage. The increase in the volume of the fermentation liquid during the addition process does not exceed 5% of the initial volume of the fermentation system.

[0016] In practice, the amino acid composition is added first to build a stable peptide chain folding protective microenvironment in the recombinant bacterial cells in advance. After a 30-minute interval, the chelated manganese ion preparation is added, which allows the bacteria to take up manganese ions under optimal physiological conditions, achieving a precise timing match between peptide chain folding protection and cofactor loading. The staged addition can avoid the two feed components from directly contacting each other in the fermentation system and causing side reactions. At the same time, the volume increase during the addition process is strictly controlled to avoid excessive dilution of nutrients in the fermentation system, maintaining the nutritional environment required for high-density bacterial growth and efficient expression of exogenous proteins.

[0017] Furthermore, in the above technical solution, the operation of adjusting and maintaining the dissolved oxygen level of the fermentation system at 20%-30% is achieved by linking the stirring speed and the gas composition introduced into the fermentation system. The gas composition is a mixture of air and pure oxygen, and the volume percentage of pure oxygen in the mixture is 10%-15%. The upper limit of the stirring speed is 600 rpm.

[0018] In practice, by adjusting the stirring speed and gas composition in a coordinated manner, the target dissolved oxygen level can be stably maintained at a lower stirring speed, avoiding mechanical damage to the recombinant bacterial cells caused by the high shear force generated by high-speed stirring. At the same time, the 10%-15% pure oxygen ratio can avoid the accumulation of intracellular reactive oxygen free radicals caused by excessive pure oxygen introduction, reduce the oxidative damage of reactive oxygen to enzyme protein amino acid residues, maintain the stability of the enzyme protein spatial conformation, and provide a stable intracellular redox environment for the correct folding of the enzyme protein.

[0019] Furthermore, in the above technical solution, after collecting the fermentation broth, the fermentation broth is subjected to cell wall disruption treatment. The cell wall disruption treatment includes adding sodium chloride, an osmotic pressure regulator, to the fermentation broth to a final concentration of 0.8-1.2 mol / L, allowing it to stand at 4°C for 30 minutes, and then using an ultrasonic disruptor under ice bath conditions to disrupt the broth. The ultrasonic disruptor operates at a frequency of 20 kHz, with a single ultrasonic operation duration of 5 seconds, a single ultrasonic interval duration of 10 seconds, and a total ultrasonic treatment duration of 20 minutes.

[0020] In practice, 0.8-1.2 mol / L sodium chloride can adjust the osmotic pressure of the fermentation broth, allowing the cell cells to undergo appropriate plasmolysis at low temperatures, reducing the mechanical strength of the cell wall and cell membrane, and significantly improving the cell disruption efficiency of subsequent ultrasonic disruption. The 4℃ ice bath environment and intermittent ultrasonic treatment can avoid thermal denaturation of enzyme proteins caused by the heat generated during ultrasonication, maximizing the preservation of the natural conformation and biological activity of enzyme proteins. At the same time, the set ultrasonic parameters can reduce the damage to the covalent structure and spatial conformation of enzyme protein molecules by ultrasonic shearing force while ensuring cell disruption efficiency.

[0021] Furthermore, in the above technical solution, the fermentation broth after the cell wall disruption treatment is subjected to solid-liquid separation, and the supernatant is purified by passing it through a nickel ion affinity chromatography column. The packing material of the nickel ion affinity chromatography column is agarose microspheres with polyethylene glycol chains grafted on their surface. Before the supernatant flows through the nickel ion affinity chromatography column, imidazole is added to the supernatant to a final concentration of 10 mmol / L. The residence time of the supernatant in the nickel ion affinity chromatography column is 5 minutes.

[0022] In practice, agarose microspheres grafted with polyethylene glycol chains can significantly reduce non-specific hydrophobic adsorption on the packing surface, reduce non-specific binding of impurities to the packing, decrease the co-elution ratio of impurities, and improve the purity of the target enzyme protein. A low concentration of 10 mmol / L imidazole can pre-block low-affinity binding sites on the chromatography column, further avoiding non-specific binding of impurities. A residence time of 5 minutes can ensure that the target allulose-3-epimerase protein with histidine tag is fully bound to the nickel ion affinity packing, thereby improving the purification and recovery rate of the target protein.

[0023] Furthermore, in the above technical solution, the eluent purified by the nickel ion affinity chromatography column is subjected to freeze-drying. Before the freeze-drying process, a freeze-drying protectant is added to the eluent. The freeze-drying protectant is a mixed aqueous solution of trehalose and mannitol. The final concentration of trehalose in the eluent is 50 g / L, and the final concentration of mannitol in the eluent is 20 g / L. The first-order sublimation temperature of the freeze-drying process is -30°C, and the second-order desorption drying temperature is 25°C.

[0024] In practice, trehalose can bind to hydrophilic groups on the surface of enzyme protein molecules through hydrogen bonds, replacing water molecules to maintain the natural spatial conformation of the enzyme protein during freeze-drying and preventing protein molecules from agglomerating and denaturing due to dehydration. Mannitol can be used as a freeze-drying filler to improve the shapeability and porosity of the freeze-dried product, while also working synergistically with trehalose to protect the conformation. The two-stage gradient temperature freeze-drying process can quickly remove free water from the system in the first sublimation stage and gently remove bound water in the second desorption drying stage, avoiding the destruction of the spatial conformation of the enzyme protein caused by sudden temperature changes, and finally obtaining a highly stable solid allulose-3-epimerase preparation.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention addresses the core problem of insufficient enzyme thermal stability in conventional fermentation processes by simultaneously supplementing a combination of arginine and glutamic acid, along with a manganese ion chelate formulation, during the late logarithmic growth phase. This is achieved by using amino acids to intervene in the microenvironment of nascent peptide chain folding, thereby inhibiting inclusion body formation caused by hydrophobic aggregation. Simultaneously, chelated manganese ions directly act as cofactor precursors, binding in situ to the active site of the enzyme protein within the cell. This addresses both the correctness of protein folding and the cofactor loading rate. Furthermore, the combined operation of adjusting the stirring speed and maintaining a specific dissolved oxygen level using a mixture of air and pure oxygen provides a suitable microenvironment for correct enzyme protein folding and metal ion embedding.

[0026] 2. By determining the timing of supplementation based on the inflection point of the rate of decrease in biomass and pH in the fermentation broth, the interference of intervention node deviations on the physiological state of the cells was avoided. The amino acid composition was prepared into sustained-release microcapsules of gelatin and sodium alginate cross-linked polymer containing L-proline. A staged, intermittent feeding strategy of the composition and chelated formulation was adopted to eliminate the osmotic pressure shock caused by excessively high local concentrations and limit the abnormal increase in fermentation broth volume. A manganese ion-ethylenediamine di-o-hydroxyphenylacetic acid complex was used as a chelating agent to block the precipitation and loss pathway of free metal ions in the fermentation broth. Osmotic pressure adjustment with sodium chloride and low-temperature settling before cell disruption, combined with purification using a nickel ion affinity chromatography column with agarose microspheres grafted with polyethylene glycol chains, reduced the co-elution ratio of impurities. Trehalose and mannitol were added to the eluent for two-stage freeze-drying, maintaining the conformation of the enzyme protein during the dehydration stage. Detailed Implementation

[0027] The recombinant bacteria containing the allulose-3-epimerase gene used in this specific embodiment is a recombinant engineered bacterium obtained by cloning the allulose-3-epimerase encoding gene from Clostridium botleae into the pET-28a(+) expression vector and transforming it into Escherichia coli BL21(DE3) competent cells. The construction method of this strain is a conventional genetic engineering operation in the art. All chemical reagents used are of biochemical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd.; ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) was purchased from Sigma-Aldrich; the nickel ion affinity chromatography packing material is Ni-IDA agarose microspheres with polyethylene glycol chains grafted on the surface, purchased from GE Healthcare; the fermenter is a 5L mechanically stirred fermenter, purchased from Shanghai Baoxing Bio-Equipment Engineering Co., Ltd.

[0028] The basal culture medium used in this specific embodiment is a modified TB medium, the components of which are: 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 2.31 g / L potassium dihydrogen phosphate, 12.54 g / L dipotassium hydrogen phosphate, pH adjusted to 7.2, and used after autoclaving at 121°C for 20 min.

[0029] Example 1: Seed culture preparation: A single colony of recombinant Escherichia coli BL21(DE3) was picked and inoculated into 50 mL of LB liquid medium. The culture was incubated at 37°C with shaking at 200 rpm for 12 h to obtain primary seed culture. The primary seed culture was then transferred to 500 mL of LB liquid medium at a 2% inoculation rate and incubated at 37°C with shaking at 200 rpm for 8 h to obtain secondary seed culture. The OD of the secondary seed culture was... 600 It is 6.0 ± 0.2.

[0030] Fermentation culture: The secondary seed culture was inoculated into a 5L mechanically stirred fermenter at a 5% inoculation rate. The basal culture medium was 3L. The initial fermentation conditions were: temperature 37℃, initial stirring speed 300rpm, aeration rate 1.5vvm, and dissolved oxygen electrode calibrated to 100% after air saturation. During the fermentation process, dissolved oxygen was linked to stirring speed to maintain a dissolved oxygen level of not less than 40%.

[0031] Feeding operation: Online monitoring of the OD of the fermentation broth 600 With pH value, when the fermentation broth OD 600 Reaching 2.7 (OD after initial inoculation) 600 When the pH value is 9 times that of 0.3, and the rate of decrease of the fermentation broth pH value decreases from 0.15h⁻¹ to 0.05h⁻¹ and an inflection point is reached, a supplementary addition operation is performed.

[0032] The sustained-release microcapsules composed of L-arginine, L-glutamic acid, and L-proline were prepared using a complex coagulation method: gelatin and sodium alginate were dissolved in deionized water at a mass ratio of 2:1 to prepare a 2% (w / v) aqueous solution for the capsule wall. A mixed aqueous solution of L-arginine, L-glutamic acid, and L-proline was added, and the mixture was stirred evenly at 35°C. 10% glacial acetic acid was added dropwise to adjust the pH to 4.0, and a complex coagulation reaction occurred to form microcapsules. Glutaraldehyde was added for cross-linking and curing for 30 min. After washing, the microcapsules were sieved, and those with a particle size of 75 μm were collected. The release half-life of the microcapsules in modified TB medium was determined to be 3 h. The mass ratio of L-arginine to L-glutamic acid was 1:2.25, and the mass ratio of L-proline to L-arginine was 1:2.

[0033] The preparation method of the chelated preparation containing divalent manganese ions is as follows: weigh manganese sulfate and EDDHA at a molar ratio of 1:1.2, dissolve them in deionized water, adjust the pH to 7.0, stir at room temperature for 2 hours to form a stable complex aqueous solution, filter it through a 0.22 μm filter membrane for sterilization, and store it at 4°C for later use.

[0034] The addition was carried out using a staged fed-batch strategy: In the first stage, the slow-release microcapsule suspension was fed at a constant flow rate using a syringe pump for 1 hour. After the addition, the final concentrations of L-arginine, L-glutamic acid, and L-proline in the basal medium were 0.6 g / L, 1.35 g / L, and 0.3 g / L, respectively. After a 30-minute interval, the second stage began, in which the chelated formulation was fed for 0.5 hours. After the addition, the final concentration of the chelated formulation in the basal medium was 1.2 mmol / L. The total increase in fermentation broth volume during the entire feeding process was 120 mL, which did not exceed 5% of the initial volume of the fermentation system.

[0035] Dissolved oxygen control: After the replenishment operation is completed, the dissolved oxygen level of the fermentation system is adjusted and maintained at 25% until the end of fermentation. Dissolved oxygen control is achieved by linking the stirring speed and the composition of the introduced gas. The gas is a mixture of air and pure oxygen, with pure oxygen accounting for 12% of the volume of the mixture. The upper limit of the stirring speed is 600 rpm.

[0036] Fermentation broth collection: Stop the culture after a total fermentation time of 24 hours and collect the fermentation broth.

[0037] Cell wall disruption treatment: Add sodium chloride, an osmotic pressure regulator, to the collected fermentation broth to a final concentration of 1.0 mol / L. After standing at 4°C for 30 minutes, use an ultrasonic disruptor under ice bath conditions to disrupt the cell wall. The ultrasonic disruptor operates at a frequency of 20 kHz, with a single ultrasonic operation lasting 5 seconds and a single ultrasonic interval lasting 10 seconds. The total ultrasonic treatment time is 20 minutes.

[0038] Purification: The fermentation broth after cell wall disruption was centrifuged at 4℃ and 8000rpm for 20min for solid-liquid separation. The supernatant was collected, and imidazole was added to the supernatant to a final concentration of 10mmol / L. The supernatant was purified by passing it through a nickel ion affinity chromatography column. The residence time of the supernatant in the chromatography column was 5min. After elution to remove impurities, the target protein eluent was collected.

[0039] Freeze-drying: A freeze-drying protectant, a mixed aqueous solution of trehalose and mannitol, was added to the eluent. After addition, the final concentration of trehalose in the eluent was 50 g / L, and the final concentration of mannitol was 20 g / L. The mixed eluent was then freeze-dried. The freeze-drying procedure was as follows: first-stage sublimation temperature -30℃, second-stage desorption drying temperature 25℃, to obtain a solid allulose-3-epimerase preparation.

[0040] Example 2: Only the final mass ratio of L-arginine to L-glutamic acid in the basal medium was adjusted to 1:1.5. All other culture conditions, feeding parameters, and post-treatment steps were completely consistent with those in Example 1.

[0041] Example 3: Only the final mass ratio of L-arginine to L-glutamic acid in the basal medium was adjusted to 1:3.0. All other culture conditions, feeding parameters, and post-treatment steps were completely consistent with those in Example 1.

[0042] Example 4: The final concentration of the chelated preparation containing divalent manganese ions in the basal culture medium was adjusted to 0.5 mmol / L. All other culture conditions, feeding parameters, and post-treatment steps were completely consistent with those in Example 1.

[0043] Example 5: The final concentration of the chelated preparation containing divalent manganese ions in the basal culture medium was adjusted to 2.0 mmol / L. All other culture conditions, feeding parameters, and post-treatment steps were completely consistent with those in Example 1.

[0044] Example 6: Only the dissolved oxygen level of the fermentation system after the addition was adjusted to 30%, and the volume ratio of pure oxygen in the mixed gas was adjusted to 15%. All other culture conditions, feeding parameters, and post-treatment steps were completely consistent with Example 1.

[0045] Example 7: Adjusting the triggering timing of the supplementation operation only to: the optical density value (OD) corresponding to the biomass of the fermentation broth. 600 When the initial inoculation light density value is 8 times and the pH value of the fermentation broth shows an inflection point in the rate of decrease, the addition of the composition and chelated preparation is performed. All other culture conditions, feeding parameters, and post-processing steps are completely consistent with those in Example 1.

[0046] Example 8: Adjusting the triggering timing of the supplementation operation only to: the optical density value (OD) corresponding to the biomass of the fermentation broth. 600 When the light density reaches 10 times the initial inoculation value and the pH value of the fermentation broth shows an inflection point in the rate of decrease, the addition of the composition and chelated preparation is performed. All other culture conditions, feeding parameters, and post-processing steps are completely consistent with those in Example 1.

[0047] Comparative Example 1: Only the addition of the L-arginine and L-glutamic acid combination was omitted; all other culture conditions, chelated formulation addition parameters, dissolved oxygen control, and post-treatment steps were completely consistent with Example 1.

[0048] Comparative Example 2: The conventional fermentation preparation process described in the background art was used, specifically as follows: the same recombinant bacteria as in Example 1 were inoculated into the same basal culture medium at the same inoculation amount and fermented at 37°C until the late logarithmic growth phase. IPTG at a final concentration of 0.5 mmol / L was added to induce the expression of exogenous protein, while free manganese sulfate solution at a final concentration of 1.2 mmol / L was added simultaneously. During fermentation, the dissolved oxygen level was maintained at no less than 30% by increasing the stirring speed (maximum 800 rpm) and aeration rate (maximum 2.0 vvm). The fermentation broth was collected after 24 hours of fermentation. The fermentation broth was directly subjected to ice bath ultrasonic disruption, with disruption parameters consistent with Example 1. After disruption, the supernatant was collected by centrifugation and purified using a conventional Ni-IDA agarose microsphere chromatography column without grafted polyethylene glycol chains. No low-concentration imidazole pretreatment was added during the purification process. The eluent was directly freeze-dried without adding a freeze-drying protectant, and the freeze-drying procedure was consistent with Example 1.

[0049] Comparative Example 3: Only the final mass ratio of L-arginine to L-glutamic acid in the basal culture medium was adjusted to 1:4.0. This ratio exceeds the range of 1:(1.5-3.0) defined in claim 1. All other culture conditions, feeding parameters, and post-processing steps were completely consistent with those in Example 1.

[0050] Comparative Example 4: The only difference was that the combination of L-arginine and L-glutamic acid was replaced with a free aqueous solution and added once, without using the sustained-release microcapsule form. All other culture conditions, final feed concentration, feeding strategy, and post-treatment steps were completely consistent with Example 1.

[0051] Test method: Cell density detection: The optical density (OD) of the fermentation broth was measured using a UV-Vis spectrophotometer at a wavelength of 600 nm. 600 , characterizing the cell growth density.

[0052] Inclusion body proportion detection: Fermentation broth cells were taken, ultrasonically disrupted, and centrifuged at 4℃ and 12000rpm for 20min. The supernatant (soluble protein) and precipitate (inclusion bodies) were collected separately. SDS-PAGE electrophoresis combined with gel imaging system was used for quantitative analysis to calculate the proportion of inclusion body protein in the total target protein.

[0053] Enzyme activity assay: The enzyme activity of allulose-3-epimerase was determined using the cysteine-carbazole method. Enzyme activity was defined as the amount of enzyme required to catalyze the production of 1 μmol of D-allulose per minute from D-fructose at pH 8.0 and 60℃, which is 1 unit of enzyme activity (U). Protein concentration was determined using the Bradford method, and specific enzyme activity (U / mg) was calculated.

[0054] Thermal stability test: The purified enzyme solution was adjusted to a protein concentration of 1 mg / mL and incubated in a 55℃ water bath for 2 hours. The enzyme activity before and after incubation was measured, and the percentage of residual enzyme activity was calculated. The higher the residual enzyme activity, the better the thermal stability.

[0055] Protein purity detection: The purity of the purified target protein was detected by high performance liquid chromatography (HPLC) combined with SDS-PAGE gel imaging system.

[0056] Total enzyme recovery rate detection: The total enzyme activity recovery rate of the final enzyme preparation after freeze-drying was calculated based on the total enzyme activity in the fermentation broth.

[0057] Test results: Table 1. Test results of fermentation preparation effects of each embodiment and comparative example. Results analysis: The technical solutions of Examples 1-8 of this invention all achieve significantly better technical effects than the comparative examples: the bacterial cell OD of the example group 600 All values ​​remained above 56, the inclusion body ratio was below 5%, the specific enzyme activity was above 400 U / mg, the residual enzyme activity after incubation at 55℃ for 2 hours was above 87%, the target protein purity was above 98%, and the total enzyme recovery rate was above 73%. This fully solves the core defects of conventional processes in the background technology, such as high inclusion body formation rate and insufficient enzyme thermal stability.

[0058] Example 1, as the best embodiment, has the best performance indicators, proving that the combination of core parameters defined in this invention has a synergistic effect and can achieve high-stability preparation of allulose-3-epimerase from multiple dimensions such as protein correct folding rate, cofactor loading rate, cell growth status, and purification recovery rate.

[0059] Comparative Example 1 lacked the addition of the L-arginine and L-glutamic acid combination, which led to a sharp increase in the inclusion body ratio to 38.7%, and a significant decrease in enzyme activity and thermal stability. This proves that the amino acid combination is the core technical feature for inhibiting peptide chain misfolding and reducing inclusion body formation, and directly determines the correct folding rate and basic activity of the enzyme protein.

[0060] Comparative Example 2 uses conventional processes of existing technology, and its inclusion body ratio is as high as 42.3%, the residual enzyme activity is only 32.7%, and the total enzyme recovery rate is less than 30%. This is significantly different from Example 1 of the present invention, which directly proves that the technical solution of the present invention has unexpected technical effects compared with the prior art and solves the technical pain points that have existed in the prior art for a long time.

[0061] Comparative Example 3 adjusted the amino acid mass ratio to outside the scope defined in the claims, resulting in inhibited cell growth, a significant increase in the proportion of inclusion bodies, and a substantial decrease in enzyme activity and thermal stability. This demonstrates that the amino acid ratio range defined in this invention is a key parameter to ensure the technical effect, and exceeding this range will prevent the achievement of the intended purpose of the invention.

[0062] Comparative Example 4 omitted the method of adding sustained-release microcapsules and directly added free amino acids in one go, which caused a sudden increase in local osmotic pressure in the fermentation system, affected cell growth, and significantly increased the inclusion body formation rate. This proves that the method of adding sustained-release microcapsules is a key technical means to maintain a continuous and effective concentration of amino acids and avoid osmotic pressure shocks in the cells, which can further improve the technical effect of the invention.

[0063] In summary, this invention achieves highly stable fermentation preparation of allulose-3-epimerase by coupling the addition of an amino acid composition with a chelated manganese ion preparation, combined with specific addition timing, feeding strategy, dissolved oxygen control, and post-treatment processes, through the synergistic effect of multiple core links such as protein folding, cofactor loading, and conformational stabilization. Compared with the prior art, this invention has outstanding substantive features and significant progress, and possesses the inventiveness required by patent law.

Claims

1. A method for preparing a highly stable allulose-3-epimerase by fermentation, comprising the step of inoculating a recombinant strain containing an allulose-3-epimerase gene into a basal culture medium for fermentation culture, characterized in that, During the late logarithmic growth phase of the recombinant bacteria fermentation, a combination of L-arginine and L-glutamic acid is added to the basal culture medium, and a chelated preparation containing divalent manganese ions is coupled into the basal culture medium. The final concentration ratio of L-arginine to L-glutamic acid in the basal culture medium is 1:(1.5-3.0), and the final concentration of the chelated preparation containing divalent manganese ions in the basal culture medium is 0.5-2.0 mmol / L. After the addition of the composition and the chelated preparation is completed, the dissolved oxygen level of the fermentation system is adjusted and maintained at 20%-30% until the fermentation endpoint is reached, and the fermentation broth is collected.

2. The method according to claim 1, characterized in that, The late logarithmic growth phase is determined by online monitoring of the biomass and pH value of the fermentation broth. When the optical density value corresponding to the biomass of the fermentation broth reaches 8-10 times the optical density value at the initial inoculation, and the pH value of the fermentation broth shows an inflection point in the rate of decrease, the addition of the composition and the chelated preparation is performed.

3. The method according to claim 1, characterized in that, The composition of L-arginine and L-glutamic acid is added in the form of sustained-release microcapsules. The capsule wall material of the sustained-release microcapsules is a cross-linked polymer of gelatin and sodium alginate. The particle size of the sustained-release microcapsules is 50-100 micrometers. The release half-life of the sustained-release microcapsules in the basal culture medium is 2-4 hours.

4. The method according to claim 3, characterized in that, The sustained-release microcapsule also contains L-proline, with a mass ratio of L-proline to L-arginine of 1:

2. The final concentration of L-proline added to the basal culture medium is 0.1-0.5 g / L. The sustained-release microcapsule is pumped into the basal culture medium at a constant flow rate using an injection pump.

5. The method according to claim 1, characterized in that, The chelated formulation containing divalent manganese ions is an aqueous solution of a complex of divalent manganese ions and ethylenediamine di-o-hydroxyphenylacetic acid, wherein the molar ratio of divalent manganese ions to ethylenediamine di-o-hydroxyphenylacetic acid in the aqueous solution is 1:1.2, and the aqueous solution is subjected to sterilization filtration through a 0.22-micron filter membrane before being added.

6. The method according to claim 5, characterized in that, The composition and the chelated preparation are added using a staged addition strategy. The composition is added in the first stage for 1 hour, and the chelated preparation is added in the second stage for 0.5 hours. There is a 30-minute interval between the first and second stages. The increase in fermentation broth volume during the addition process does not exceed 5% of the initial volume of the fermentation system.

7. The method according to claim 1, characterized in that, The operation of adjusting and maintaining the dissolved oxygen level of the fermentation system at 20%-30% is achieved by linking the stirring speed and the gas composition introduced into the fermentation system. The gas composition is a mixture of air and pure oxygen, with the volume percentage of pure oxygen in the mixture being 10%-15%, and the upper limit of the stirring speed adjustment is 600 rpm.

8. The method according to claim 7, characterized in that, After collecting the fermentation broth, the fermentation broth is subjected to cell wall disruption treatment. The cell wall disruption treatment includes adding sodium chloride, an osmotic pressure regulator, to the fermentation broth to a final concentration of 0.8-1.2 mol / L, allowing it to stand at 4°C for 30 minutes, and then using an ultrasonic disruptor under ice bath conditions to disrupt the broth. The ultrasonic disruptor operates at a frequency of 20 kHz, with a single ultrasonic operation lasting 5 seconds, a single ultrasonic interval lasting 10 seconds, and a total ultrasonic treatment time of 20 minutes.

9. The method according to claim 8, characterized in that, The fermentation broth after the cell wall disruption treatment was subjected to solid-liquid separation, and the supernatant was purified by passing it through a nickel ion affinity chromatography column. The packing material of the nickel ion affinity chromatography column was agarose microspheres with polyethylene glycol chains grafted on their surface. Before the supernatant flowed through the nickel ion affinity chromatography column, imidazole was added to the supernatant to a final concentration of 10 mmol / L. The residence time of the supernatant in the nickel ion affinity chromatography column was 5 minutes.

10. The method according to claim 9, characterized in that, The eluent purified by the nickel ion affinity chromatography column was freeze-dried. Before the freeze-drying process, a freeze-drying protectant was added to the eluent. The freeze-drying protectant was a mixed aqueous solution of trehalose and mannitol. The final concentration of trehalose in the eluent was 50 g / L, and the final concentration of mannitol in the eluent was 20 g / L. The first sublimation temperature of the freeze-drying process was -30°C, and the second-stage desorption drying temperature was 25°C.

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