A method of preparing proline amide catalyzed by a carboxylate reductase

Abstract

The application relates to the technical field of biological catalysis, and discloses a method for preparing prolinamide by carboxylic acid reductase catalysis, which comprises the following steps: adding pure water, basic buffer salt, polyethylene glycol 400, an initial carbon source and a recombinant whole-cell biological catalyst into a reaction kettle, mixing to form an activation system, and stirring and aeration; after cooling, injecting a phase transition trigger into the kettle, triggering mesophase separation through a sharp increase in salt concentration, and converting the activation system into a microemulsion state; continuously cooling to a constant temperature, starting double-channel feeding, dynamically supplementing the carbon source in channel A to maintain a low residual concentration, and dropping a mixed solution of L-proline and ammonium salt in channel B; and collecting product liquid after reaction. According to the application, a hydrophobic wrapping layer is formed on the cell surface by polyethylene glycol to limit free water penetration, and an intermediate hydrolysis path is cut off; meanwhile, stepwise cooling and limited carbon source supply are used to inhibit byproduct accumulation and maintain energy regeneration stability, so that the substrate conversion rate is improved.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, specifically to a method for preparing proline catalyzed by carboxylic acid reductase. Background Technology

[0002] Proline amide, as an important chiral structural unit, has wide applications in pharmaceutical synthesis and fine chemical engineering. Industrial production utilizes chemical synthesis, primarily through the esterification and ammonolysis of proline. Currently, there are no reports of de novo biosynthesis of proline amide, and enzymatic synthesis is limited to lipases, which suffer from low catalytic efficiency. Therefore, the discovery of new enzymes, the design of catalytic reactions, and the development of key technologies are needed.

[0003] Based on the principle of ATP activation, the synthesis of prolineamide through the ammonolysis of L-proline with an ammonia source using a recombinant whole-cell biocatalyst containing carboxyl reductase and phosphoadenosine transferase holds significant development potential. However, in conventional homogeneous aqueous reaction systems, this whole-cell ammonolysis catalysis process faces the technical bottleneck of low substrate conversion rates. During the intracellular enzymatic reaction, L-proline is activated to a prolyl-AMP intermediate under the energy supply of adenosine triphosphate (ATP), and this intermediate is then subjected to ammonolysis to generate the final product. Due to the presence of a large number of free water molecules in conventional reaction systems, this highly reactive intermediate is subjected to nucleophilic attack by water molecules and undergoes hydrolysis, reverting to the initial substrate L-proline. This spontaneous side reaction leads to severe substrate inefficiency cycling, hindering the accumulation of the target product.

[0004] In conventional processes that attempt to extend the catalytic reaction cycle to increase yield, it is difficult to maintain metabolic and energy homeostasis in the chassis cells. Conventional isothermal operations and extensive substrate addition methods easily lead to a shift in the cell's carbon metabolism towards the fermentation pathway, resulting in the rapid accumulation of weakly acidic byproducts such as acetic acid in the fermentation broth. This accumulation of organic acids disrupts the transmembrane proton gradient across the cell membrane, blocking the transmembrane energy regeneration cycle, ultimately leading to the depletion of intracellular adenosine triphosphate (ATP) reserves and metabolic function decline, causing the long-cycle catalytic reaction to be unable to continue in its later stages. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing proline amide by carboxylic acid reductase catalysis. This method solves the problems of high-energy intermediates being easily attacked by free water molecules and undergoing hydrolysis in conventional aqueous reaction systems, leading to severe substrate ineffective cycling, as well as the low conversion rate of the target product and the difficulty in sustaining the reaction due to the accumulation of toxic byproducts and depletion of intracellular energy during long-cycle whole-cell catalysis.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for preparing proline catalyzed by carboxylic acid reductase includes the following steps: Pure water, basic buffer salt, polyethylene glycol 400, initial carbon source and recombinant whole-cell biocatalyst were added to the reactor and mixed evenly to form a homogeneous activation system. At the first temperature, the activation system is aerated and stirred; Turn on the cooling system. When the activation system cools down to the second temperature, inject a phase change trigger into the reactor. The sudden increase in salt concentration triggers mesoscopic phase separation, causing the activation system to transform into a microemulsion state reaction system. The reaction system was cooled down and kept constant at the third temperature. The dual-channel feed was started. Channel A continuously added carbon source and dynamically adjusted to maintain the concentration of residual carbon source in the reactor below the set threshold. Channel B continuously added a mixed aqueous solution of L-proline and ammonium salt at a uniform rate. After the reaction system has completed, collect the product liquid containing proline amide.

[0008] By adopting the above technical solution, this invention blocks the hydrolysis pathway of intermediates and maintains cellular energy homeostasis by controlling the phase change microenvironment reconstruction and spatiotemporal metabolic decoupling, thereby improving product conversion rate.

[0009] During the reaction, the phosphoadenosine transferase within the recombinant whole-cell biocatalyst is first activated. Powered by adenosine triphosphate (ATP), it drives carboxylic acid reductase to catalyze the formation of the highly reactive prolyl-AMP intermediate from the substrate L-proline. This intermediate then undergoes ammonolysis with free ammonia in the liquid phase, generating the target products proline and adenosine monophosphate (ATP). However, in conventional homogeneous aqueous reactions, due to nucleophilic attack by bulk water molecules, the prolyl-AMP intermediate undergoes irreversible hydrolysis and reverts to L-proline, resulting in the ineffective consumption of substrate and energy.

[0010] To address the aforementioned competitive hydrolysis reaction, this invention introduces a phase transition intervention mechanism. A phase transition trigger is injected into the system containing polyethylene glycol 400, altering the original thermodynamic equilibrium and initiating competitive hydration. At this point, the solubility of polyethylene glycol 400 decreases, leading to self-assembly, sedimentation, and attachment to the surface of the engineered bacterial cells, causing the system to transform from a homogeneous solution to a microemulsion state. This polymer network surrounding the cells forms a hydrophobic barrier; its pore size and properties allow small molecules such as L-proline and ammonia sources to diffuse inward, while simultaneously restricting the disordered permeation of bulk water molecules into the intracellular enzyme active site, thus interrupting the reaction pathway where intermediates are attacked by water molecules.

[0011] To support the long-cycle catalytic reaction, cells require continuous energy regeneration. During the aeration activation phase, chassis cells accumulate a large amount of adenosine triphosphate (ATP) through oxidative phosphorylation. Upon entering the fed-batch catalysis phase, the reaction system undergoes stepwise cooling, reducing the basal maintenance energy consumption of the cells. Simultaneously, through the dynamic regulation of channel A, the residual carbon source is maintained at an extremely low threshold. This substrate-limited state inhibits the deflection of carbon metabolism towards the fermentation pathway, avoiding the formation of weakly acidic byproducts such as acetic acid. This maintains the transmembrane proton gradient across the cell membrane, allowing the limited carbon source to be directed to the energy regeneration cycle, ensuring a continuous supply of intracellular energy required for ammonolysis.

[0012] Preferably, polyethylene glycol 400 accounts for 4wt% to 10wt% of the total mass of the activation system; the initial carbon source is glucose, and the initial concentration of the initial carbon source in the activation system is 1.0 g / L to 3.0 g / L; the cell concentration of the recombinant whole-cell biocatalyst in the activation system reaches OD. 600 It ranges from 15 to 25.

[0013] By employing the above technical solution, polyethylene glycol 400, as the macromolecular backbone, can form a condensed coating layer around the cell during phase transition. The predetermined ratio of initial carbon source concentration to cell density allows the cell to complete the synthesis of energy substances during the activation phase, while avoiding carbon source enrichment in the early stages of the reaction.

[0014] A preferred method for preparing the recombinant whole-cell biocatalyst is as follows: A recombinant expression plasmid containing a carboxylic acid reductase gene from *Mycobacterium tumefaciens* and a phosphoadenosyltransferase gene from *Bacillus subtilis* is transformed into competent *Escherichia coli* cells to obtain a positive recombinant engineered strain; the positive recombinant engineered strain is inoculated into a fermentation medium, and isopropyl-β-D-thiogalactoside is added at a final concentration of 0.05 mmol / L to 0.2 mmol / L at a temperature of 16℃ to 30℃ and cultured for 16 to 24 hours to obtain a fermentation broth; the wet bacterial cells precipitated in the fermentation broth are collected by centrifugation to obtain the recombinant whole-cell biocatalyst.

[0015] By employing the above-described technical solution, a co-expression system containing relevant genes provides the enzyme combination required for the target reaction. Low-temperature conditions combined with the use of low-concentration inducers reduce the rate of intracellular protein synthesis, which decreases post-translational peptide mismatches and inclusion body formation, making the recombinase more likely to be expressed in a soluble form.

[0016] Preferably, the competent Escherichia coli cells are Escherichia coli BL21(DE3); the recombinant expression plasmid is pETDuet-1 recombinant expression plasmid.

[0017] By adopting the above technical solution, using the T7 RNA polymerase expression chassis and co-expression vector, the co-transcription of carboxylic acid reductase and phosphoadenosine transferase in the same host cell was realized, providing a catalytic basis for the ammonolysis reaction.

[0018] Preferably, the specific composition and ratio of the basic buffer salt in the activation system are limited to: MgSO4 concentration of 0.1 g / L to 0.5 g / L; The concentration of Na2HPO4·12H2O is 10.0 g / L to 20.0 g / L; The concentration of KH2PO4 is 1.0 g / L to 5.0 g / L; The concentration of NaCl is 0.1 g / L to 1.0 g / L.

[0019] By adopting the above technical solution, the above phosphate system provides an ionic environment for cells to maintain the osmotic pressure balance inside and outside the membrane, and its acid-base buffer capacity can reduce the fluctuation of microenvironment acidity and alkalinity caused by ammonia metabolism during fed-batch reaction.

[0020] Preferably, the first temperature is 32℃~37℃, the stirring speed is 150rpm~250rpm, and the operation is maintained for 1.0h~2.0h.

[0021] By adopting the above technical solution, the temperature and oxygen supply conditions are matched to the physiological needs of aerobic metabolism in chassis cells, and the high mass transfer efficiency promotes the operation of the cellular respiratory chain to complete the construction of the early high-energy molecular pool.

[0022] Preferably, the second temperature is 28℃~29℃; the phase change trigger is anhydrous sodium sulfate, and the phase change trigger is pumped into the reactor instantaneously within 3min~10min through a high-pressure metering pump, so that the final concentration of the phase change trigger in the reaction system rapidly reaches 30.0g / L~60.0g / L.

[0023] By employing the above technical solution, injecting a high concentration of anhydrous sodium sulfate into the system causes a rapid increase in osmotic pressure, leading to hydration of inorganic salt ions and free water molecules. The original polymer network loses its hydration layer, resulting in mesophase separation and the precipitation of a hydrophobic layer outside the cells.

[0024] Preferably, the third temperature is 22℃~26℃; in channel B, the total added amount of L-proline and ammonium salt, converted to the concentration in the reaction system, are: L-proline 8.0g / L~15.0g / L, and ammonium salt is ammonium chloride with a concentration of 4.0g / L~8.0g / L; the addition time of channel B lasts for 8h~14h.

[0025] By employing the above-mentioned technical solution, the substrate is added at a uniform rate within the cooling range, mitigating the metabolic load and osmotic pressure changes caused by instantaneously high substrate concentrations. The substrate input rate is adapted to the consumption rate of the intracellular enzymatic reaction, maintaining the continuity of the catalytic reaction.

[0026] Preferably, during the dual-channel feeding process, channel A is dynamically adjusted according to the carbon source consumption rate; the threshold is set to 0.5 g / L to 1.0 g / L.

[0027] By employing the above-mentioned technical solution, a low level of residual carbon source concentration was maintained, suppressing fermentation side reactions such as substrate-level phosphorylation. This reduced the production of organic acids, avoided disruption of the transmembrane proton gradient, and ensured the normal operation of the adenosine triphosphate (ATP) synthesis pathway.

[0028] Preferably, the steps for collecting the product liquid containing proline amide include: adding 1 to 2 times the volume of deionized water to the reaction system after the reaction is completed to dilute and demulsify to restore homogeneity, then centrifuging to remove the bacterial cells, and collecting the supernatant to obtain the product liquid containing proline amide.

[0029] By adopting the above technical solution, the addition of deionized water reduces the salt concentration of the system, reversing the phase separation state of polyethylene glycol 400. The reaction solution is restored to a low-viscosity homogeneous state, thereby improving the solid-liquid separation effect of subsequent centrifugation sterilization.

[0030] This invention provides a method for preparing proline catalyzed by carboxylic acid reductase. It has the following beneficial effects: 1. This invention triggers mesoscopic phase separation in a reaction system containing polyethylene glycol 400 by instantaneously injecting anhydrous sodium sulfate. Due to its decreased solubility, polyethylene glycol settles on the surface of engineered bacterial cells, forming a coating layer. While allowing substrates such as ammonia to diffuse inward, this restricts the penetration of bulk free water molecules into the intracellular enzyme active site, blocking the spontaneous hydrolysis of the prolyl-AMP intermediate in a water-rich environment, reducing ineffective substrate recycling, and improving the final conversion rate of the target product, prolyl amide.

[0031] 2. This invention employs a reaction control scheme combining stepped cooling and dual-channel dynamic fed-batch processing. Lowering the temperature after entering the fed-batch catalysis stage reduces the basal maintenance energy consumption of the cell; simultaneously, dynamically adjusting the carbon source replenishment amount maintains an extremely low residual carbon source concentration, inhibiting the shift of carbon metabolism towards the fermentation pathway that produces byproducts such as acetic acid, avoiding the destruction of the transmembrane proton gradient by the accumulation of weak acids, and directing the carbon source to the transmembrane energy regeneration cycle, thus ensuring a stable supply of intracellular adenosine triphosphate (ATP) during long-cycle catalysis.

[0032] 3. This invention introduces a post-treatment step of water dilution and demulsification after the catalytic reaction. By adding deionized water to the microemulsion system, the concentration of inorganic salts in the environment is reduced, reversing the phase separation state of polyethylene glycol 400, restoring the reaction solution to a low-viscosity homogeneous aqueous solution, eliminating the obstruction of product separation by the polymer aggregate layer, improving the solid-liquid separation efficiency of the centrifugation sterilization process, and facilitating the rapid collection of the product liquid. Attached Figure Description

[0033] Figure 1 This is a graph showing the change in optical density of the reaction system of the present invention over time. Figure 2 This is a graph showing the change in dissolved oxygen saturation over time in the reaction system of this invention. Figure 3 This is a comparison of liquid chromatograms of the target products in different reaction systems of the present invention. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the preparation examples, examples, comparative examples, and test examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Preparation Example 1: This preparation example provides a method for preparing a recombinant whole-cell biocatalyst, comprising the following steps: Positive recombinant engineered strains were obtained by transforming the pETDuet-1 recombinant expression plasmid containing the carboxyl reductase gene from Mycobacterium marineum and the phosphoadenosyltransferase gene from Bacillus subtilis into Escherichia coli BL21(DE3) competent cells.

[0036] The positive recombinant engineered strain was inoculated into a fermentation medium and induced to grow for 20 h by adding isopropyl-β-D-thiogalactoside at a final concentration of 0.1 mmol / L at 20 °C.

[0037] The cultured fermentation broth was centrifuged at 8000 rpm for 10 min at 4°C, and the precipitated wet bacterial cells were collected to obtain the recombinant whole-cell biocatalyst.

[0038] Preparation Example 2: This preparation example provides a method for preparing a recombinant whole-cell biocatalyst, comprising the following steps: Positive recombinant engineered strains were obtained by transforming the pETDuet-1 recombinant expression plasmid containing the carboxyl reductase gene from Mycobacterium marineum and the phosphoadenosyltransferase gene from Bacillus subtilis into Escherichia coli BL21(DE3) competent cells.

[0039] The positive recombinant engineered strain was inoculated into a fermentation medium and induced to culture for 24 h with isopropyl-β-D-thiogalactoside at a final concentration of 0.05 mmol / L under conditions of 16 °C.

[0040] The cultured fermentation broth was centrifuged at 8000 rpm for 10 min at 4°C, and the precipitated wet bacterial cells were collected to obtain the recombinant whole-cell biocatalyst.

[0041] Preparation Example 3: This preparation example provides a method for preparing a recombinant whole-cell biocatalyst, comprising the following steps: Positive recombinant engineered strains were obtained by transforming the pETDuet-1 recombinant expression plasmid containing the carboxyl reductase gene from Mycobacterium marineum and the phosphoadenosyltransferase gene from Bacillus subtilis into Escherichia coli BL21(DE3) competent cells.

[0042] The positive recombinant engineered strain was inoculated into the fermentation medium, and induced to grow for 16 h by adding isopropyl-β-D-thiogalactoside at a final concentration of 0.2 mmol / L under the condition of 30℃.

[0043] The cultured fermentation broth was centrifuged at 8000 rpm for 10 min at 4°C, and the precipitated wet bacterial cells were collected to obtain the recombinant whole-cell biocatalyst.

[0044] Example 1: This embodiment provides a method for preparing proline catalyzed by carboxylic acid reductase, comprising the following steps: Pure water and basic buffer salts were added to a reaction vessel equipped with a temperature control jacket, a dual-channel feed metering pump, and a ventilated stirring system to make the concentrations of MgSO4, Na2HPO4·12H2O, KH2PO4, and NaCl in the reaction system 0.24 g / L, 15.12 g / L, 3.0 g / L, and 0.5 g / L, respectively.

[0045] Polyethylene glycol 400 (7 wt% of the total mass) and glucose (2.0 g / L as the initial carbon source) were added to the activation system. After thorough mixing, the recombinant whole-cell biocatalyst prepared in Preparation Example 1 was inoculated to bring the cell concentration of the activation system to OD0.05. 600 The value is 20, at which point the activated system is homogeneous and contains no substrate.

[0046] Turn on the agitator in the reactor, set the speed to 200 rpm and introduce sterile air. Control the temperature inside the reactor to be constant at the first temperature of 35°C by jacket heating and maintain operation for 1.5 hours.

[0047] Subsequently, the cooling system was activated for step-by-step cooling. When the temperature inside the reactor accurately dropped to the second temperature of 28°C, anhydrous sodium sulfate, which serves as a phase change trigger, was instantaneously pumped into the reactor within 5 minutes using a high-pressure metering pump, so that its final concentration in the reaction system rapidly reached 45.0 g / L. As the salt concentration increased sharply, the system underwent mesoscopic phase separation and transformed into a microemulsion state.

[0048] The system temperature continued to drop and remained constant at the third temperature of 25°C. The dual-channel feed was started, and glucose equivalent to 18.0 g / L was continuously added as a supplementary carbon source in channel A at a uniform rate. The concentration of residual sugar (i.e. residual carbon source) in the reactor was dynamically adjusted according to the consumption rate to keep it always less than the set threshold of 0.5 g / L. Channel B prepared a mixed aqueous solution of L-proline (equivalent to 10.0 g / L) and ammonium chloride (equivalent to 6.0 g / L) as an ammonium salt, and added it dropwise at a uniform rate for 12 hours.

[0049] The reaction was terminated after the stirring speed was maintained at 200 rpm for a total reaction time of 16 h. The reaction solution was diluted with one volume of deionized water to break the emulsion and restore homogeneity. Then, the solution was centrifuged at 10,000 rpm for 10 min to remove the bacterial cells. The supernatant was collected to obtain the product solution containing proline amide.

[0050] Example 2: This embodiment provides a method for preparing proline catalyzed by carboxylic acid reductase, comprising the following steps: Pure water and basic buffer salts were added to a reactor equipped with a temperature control jacket, a dual-channel feed metering pump, and a ventilated stirring system to make the concentrations of MgSO4, Na2HPO4·12H2O, KH2PO4, and NaCl in the reaction system 0.1 g / L, 10.0 g / L, 1.0 g / L, and 0.1 g / L, respectively.

[0051] Polyethylene glycol 400 (4 wt% of the total mass) and glucose (1.0 g / L as the initial carbon source) were added to the activation system. After thorough mixing, the recombinant whole-cell biocatalyst prepared in Preparation Example 2 was inoculated to bring the cell concentration of the activation system to OD0.05. 600 It is 15.

[0052] Turn on the agitator in the reactor, set the speed to 150 rpm and introduce sterile air, control the temperature inside the reactor to be constant at the first temperature of 32℃, and maintain operation for 1.0 h.

[0053] The cooling system was then activated to lower the temperature. When the temperature inside the reactor dropped to the second temperature of 29°C, anhydrous sodium sulfate, which serves as a phase change trigger, was pumped into the reactor instantaneously within 3 minutes using a high-pressure metering pump, so that its final concentration in the reaction system quickly reached 30.0 g / L to trigger the phase change.

[0054] The system temperature continued to drop and remained constant at the third temperature of 22°C. The dual-channel feed was started, and glucose equivalent to 14.0 g / L was continuously added at a constant rate through channel A as a supplementary carbon source to maintain the concentration of residual sugar (i.e. residual carbon source) in the reactor below the set threshold of 0.5 g / L. Channel B prepared a mixed aqueous solution of 8.0 g / L L-proline and 4.0 g / L ammonium chloride as an ammonium salt, and added it dropwise at a uniform rate for 8 hours.

[0055] The reaction was terminated after a total reaction time of 16 hours. The reaction solution was diluted with one volume of deionized water to break the emulsion. The solution was centrifuged at 10,000 rpm for 10 minutes to remove the bacterial cells. The supernatant was collected to obtain the product solution containing proline.

[0056] Example 3: This embodiment provides a method for preparing proline catalyzed by carboxylic acid reductase, comprising the following steps: Pure water and basic buffer salts were added to a reaction vessel equipped with a temperature control jacket, a dual-channel feed metering pump, and a ventilated stirring system to make the concentrations of MgSO4, Na2HPO4·12H2O, KH2PO4, and NaCl in the reaction system 0.5 g / L, 20.0 g / L, 5.0 g / L, and 1.0 g / L, respectively.

[0057] Polyethylene glycol 400 (10 wt% of the total mass) and glucose (3.0 g / L as the initial carbon source) were added to the activation system. After thorough mixing, the recombinant whole-cell biocatalyst prepared in Preparation Example 3 was inoculated to bring the cell concentration of the activation system to OD0.05. 600 It is 25.

[0058] Turn on the agitator in the reactor, set the speed to 250 rpm and introduce sterile air, control the temperature inside the reactor to be constant at the first temperature of 37°C, and maintain operation for 2.0 hours.

[0059] The cooling system was then activated to lower the temperature. When the temperature inside the reactor dropped to the second temperature of 29°C, anhydrous sodium sulfate, which serves as a phase change trigger, was pumped into the reactor instantaneously within 10 minutes using a high-pressure metering pump, so that its final concentration in the reaction system quickly reached 60.0 g / L to trigger the phase change.

[0060] The system temperature continued to drop and remained constant at the third temperature of 26°C. The dual-channel feed was started, and glucose equivalent to 22.0 g / L was continuously added at a constant rate through channel A as a supplementary carbon source to maintain the concentration of residual sugar (i.e. residual carbon source) in the reactor below the set threshold of 1.0 g / L. Channel B prepared a mixed aqueous solution of L-proline (equivalent to 15.0 g / L) and ammonium chloride (equivalent to 8.0 g / L) as an ammonium salt, and added it dropwise at a uniform rate for 14 hours.

[0061] The reaction was terminated after a total reaction time of 24 hours. The reaction solution was diluted with twice the volume of deionized water to break the emulsion. The solution was centrifuged at 10,000 rpm for 10 minutes to remove the bacterial cells. The supernatant was collected to obtain the product solution containing proline amide.

[0062] Example 4: This embodiment provides a method for preparing proline catalyzed by carboxylic acid reductase, comprising the following steps: Pure water and basic buffer salts were added to a reaction vessel equipped with a temperature control jacket, a dual-channel feed metering pump, and a ventilated stirring system to make the concentrations of MgSO4, Na2HPO4·12H2O, KH2PO4, and NaCl in the reaction system 0.24 g / L, 15.12 g / L, 3.0 g / L, and 0.5 g / L, respectively.

[0063] Polyethylene glycol 400 (8 wt% of the total mass) and glucose (2.0 g / L as the initial carbon source) were added to the activation system. After thorough mixing, the recombinant whole-cell biocatalyst prepared in Preparation Example 1 was inoculated to bring the cell concentration of the activation system to OD0.05. 600 It is 20.

[0064] Turn on the agitator in the reactor, set the speed to 200 rpm and introduce sterile air, control the temperature inside the reactor to be constant at the first temperature of 35℃, and maintain operation for 1.5 hours.

[0065] The cooling system was then activated to lower the temperature. When the temperature inside the reactor dropped to the second temperature of 28°C, anhydrous sodium sulfate, which serves as a phase change trigger, was pumped into the reactor instantaneously within 5 minutes using a high-pressure metering pump, so that its final concentration in the reaction system quickly reached 50.0 g / L to trigger the phase change.

[0066] The system temperature continued to drop and remained constant at the third temperature of 25°C. The dual-channel feed was started, and glucose equivalent to 23.0 g / L was continuously added at a constant rate through channel A as a supplementary carbon source to maintain the concentration of residual sugar (i.e. residual carbon source) in the reactor below the set threshold of 1.0 g / L. Channel B prepared a mixed aqueous solution of L-proline (equivalent to 12.0 g / L) and ammonium chloride (equivalent to 7.0 g / L) as an ammonium salt, and added it dropwise at a uniform rate for 10 hours.

[0067] The reaction was terminated after a total reaction time of 20 hours. The reaction solution was diluted with 1.5 times the volume of deionized water to break the emulsion. The solution was centrifuged at 10,000 rpm for 10 minutes to remove the bacterial cells. The supernatant was collected to obtain the product solution containing proline.

[0068] Comparative Example 1: Compared to Example 1, the differences are as follows: polyethylene glycol 400 is not added to the initial system; the reaction is kept at a constant temperature of 30°C throughout, without step cooling; anhydrous sodium sulfate is not injected for phase transition triggering; dual-channel addition is not used, and glucose (total 20 g / L), L-proline (total 10 g / L), and ammonium chloride (total 6 g / L) are all added to the reactor in the initial stage. All other aspects are the same.

[0069] Comparative Example 2: The difference from Example 1 is that when the temperature inside the reactor drops to 28°C, anhydrous sodium sulfate is not pumped in, meaning no phase change trigger is added throughout the reaction. Everything else is the same.

[0070] Comparative Example 3: The difference from Example 1 is that polyethylene glycol 400 is not added to the initial reaction system. Everything else is the same.

[0071] Comparative Example 4: Compared to Example 1, the differences are: the reaction temperature was kept constant at 35°C throughout, without any step cooling operation; and after 1.5 hours of reaction, anhydrous sodium sulfate was directly and instantaneously pumped into the reactor to trigger a phase transition. All other aspects remained the same.

[0072] Comparative Example 5: Compared to Example 1, the difference is that the dual-channel steady-state feed operation was not performed; instead, the remaining glucose (equivalent to 18.0 g / L), L-proline (equivalent to 10.0 g / L), and ammonium chloride (equivalent to 6.0 g / L) were added to the reactor all at once when the temperature dropped to 25°C. Everything else was the same.

[0073] Test Example 1: This test case is used to verify the phase transition response characteristics and substrate metabolic pathway distribution in the reaction system. The specific experimental steps are as follows: The reaction systems of Example 1, Comparative Example 2, and Comparative Example 3 were selected as test subjects. Starting at the end of the first cooling stage of the reaction process (10 minutes before the reactor temperature dropped to 28°C), the absorbance of the reaction solution at a wavelength of 600 nm was continuously monitored at 2-minute intervals using an online flow cell supplemented with a UV-Vis spectrophotometer. During this period, the turbidity evolution trajectory of Example 1 and Comparative Example 3 before and after salt injection was recorded, while samples for Comparative Example 2 were taken at the original time points.

[0074] After the third stage of fed-batch reaction is completed and the reaction is terminated, the reaction solutions of the above three systems are taken and centrifuged for sterilization, and the supernatant is collected.

[0075] The concentrations of residual L-proline and proline amide generated in the supernatant were determined by liquid chromatography. Material balance was performed based on the cumulative total molar amount of L-proline in each system to calculate the effective molar conversion rate of the target product. The proportion of substrate consumed but not converted into product was defined as the intermediate ineffective cycle rate.

[0076] The test results are detailed in the table below: Table 1. Phase transition characteristics and substrate metabolic pathway allocation data of the reaction systems in Example 1 and the comparative examples.

[0077] Conclusion Analysis: Combining Table 1 and Figure 1 It can be seen that, Figure 1 The optical density (OD) values ​​of each system within the process monitoring nodes (h) from 0 to 16 h during the reaction process are displayed visually. 600 Evolution trajectory. In Example 1, after crossing the initial salt injection critical point, the optical density parameter captured by its detection system experienced a dramatic nonlinear jump, rapidly climbing from the baseline level and... Figure 1 A high-level platform has been formed and maintained for a long time (according to precise measurements in Table 1, its local peak value is as high as 48.74).

[0078] The basic turbidity of high-density fermentation systems typically remains within a stable, narrow range. This sudden, explosive change in light scattering intensity, occurring throughout the entire process cycle, objectively reflects the fundamental reconstruction of the phase state within the reactor. The instantaneous injection of high-concentration sodium sulfate disrupts the original thermodynamic equilibrium. Its strong water-removing effect forces the polyethylene glycol network to lose its solubility, leading to self-assembly, sedimentation, and tight adhesion to the host cell surface, completing the phase transition from a homogeneous solution to a microemulsion. In contrast, control group 2, lacking the osmotic pressure impact of sodium sulfate, and control group 3, lacking the support of a large polymer backbone, exhibited only a limited optical density curve throughout the entire process monitoring period. Figure 1 The baseline level showed slight fluctuations, which failed to induce a mesoscopic condensation phase transition state, and the free water molecules still completely enveloped the engineered bacteria.

[0079] This long-term stable microenvironment reconstruction directly determines the metabolic trajectory of intracellular biochemical reactions. According to the substrate distribution data for each system at the catalytic endpoint in Table 1, the effective conversion rate (product) of the target substrate in Example 1 reached as high as 91.23%, while the ineffective recycling rate (intermediate hydrolysis) of consumed but not product-generated products was significantly reduced to only 3.42%. The macromolecular condensed phase surrounding the cell periphery constructs a selective hydrophobic barrier, ensuring smooth diffusion and penetration of small-molecule ammonia sources while extremely tightly blocking the disordered permeation of bulk free water into the intracellular enzyme active site.

[0080] The highly active prolyl-AMP intermediate is thus protected from water molecule attack and can successfully undergo ammonolysis to generate the final product. In Comparative Examples 2 and 3, the biocatalytic process was disrupted because this barrier was not established. A large amount of activated intermediate was directly exposed to the water-rich environment and underwent spontaneous hydrolysis, forcing the substrate to revert to its initial form and causing energy pool depletion.

[0081] Table 1 data clearly shows that the ineffective recycling rates (intermediate hydrolysis) for both are as high as 63.85% and 55.21%, respectively, while the corresponding effective conversion rates (products) drop to 24.31% and 31.88%. The phase change response mechanism cuts off the path of hydrolysis side reactions, providing a solid foundation for the implementation of aqueous phase ammonolysis catalysis.

[0082] Test Example 2: This test case was used to verify the intervention effect of the spatiotemporal metabolic decoupling mechanism on the energy metabolism network and the accumulation of by-product toxicity during long-term whole-cell catalysis. The specific experimental steps are as follows: The reaction systems of Example 1, Comparative Example 4, and Comparative Example 5 were selected as the objects of continuous monitoring and sampling analysis.

[0083] Sampling was performed at three key points in the reaction process: the end of the initial activation phase (1.5 h), the middle of the catalytic process (8 h), and the reaction termination point (16 h). At each point, 5 mL of fermentation broth was accurately aspirated and immediately placed in an ice bath to quench and terminate the metabolic reaction.

[0084] The quenched samples were centrifuged at low temperature to separate the bacterial cells from the supernatant. For the collected bacterial precipitate, intracellular metabolites were extracted using liquid nitrogen grinding and disruption. The absolute content of intracellular adenosine triphosphate (ATP) was determined using a luciferase-based chemiluminescence assay kit, and the data were standardized to intracellular ATP concentration parameters based on the cell dry weight of the samples.

[0085] The supernatant obtained by centrifugation was used to determine the cumulative concentration of acetic acid, a byproduct, in the reaction solution using a gas chromatograph equipped with a flame ionization detector.

[0086] The quantitative test results of the above biochemical indicators are detailed in the table below: Table 2. Monitoring data on energy metabolism and by-product accumulation in the examples and comparative examples

[0087] Conclusion Analysis: Combine Table 2 and Figure 2 It can be seen that, Figure 2 This study visually demonstrates the continuous dynamic evolution trajectory of dissolved oxygen saturation (%) for each test subject within a reaction time (h) ranging from 0 to 16 h. A close response relationship exists between the internal energy homeostasis of the whole-cell catalytic system and the operational state of the underlying respiratory chain, and this relationship is controlled by the chemical intervention of the temperature field and substrate supply strategy.

[0088] During the initial activation phase from 0 to 1.5 h, the dissolved oxygen curves of all test systems showed a steep downward trend, indicating that the chassis cells underwent an extremely active oxidative phosphorylation process driven by the initial substrate at 35 °C. The intracellular ATP concentration of up to 18.24 μmol / gDCW at the 1.5 h node in Table 2 confirms the successful accumulation of this early high-energy molecular pool, laying the foundation for the subsequent high-energy-consuming amidation reaction.

[0089] Cross Figure 2 After the clearly marked cooling and feed start point (i.e., the 1.5-hour process node), different operating strategies led to drastic differentiation of the biochemical microenvironment. In Example 1, by performing a step-down cooling to 25°C and simultaneously switching to dual-channel restricted feed, the dissolved oxygen curve smoothly converged and remained in a steady-state range of around 40% for a long period. This reflects that the cell's electron transport chain has entered a highly controlled and stable service period.

[0090] The extremely low-level dynamic residual sugar pressing strategy effectively blocked the pathway of carbon metabolism deflection towards overflow fermentation, firmly suppressing the concentration of the byproduct acetic acid at the 16-hour endpoint to a non-toxic baseline of 0.43 g / L. The low-temperature environment simultaneously reduced the consumption of useless basal maintenance energy in cells, allowing a very small amount of fed-batch carbon source to be precisely directed to the transmembrane energy regeneration cycle, supporting the ATP concentration in Example 1 to remain at a sufficient level of 11.27 μmol / g DCW even at the long-cycle catalytic endpoint.

[0091] The control system, lacking this tight decoupling control, inevitably collapsed physiologically and metabolically. Comparative Example 4 (at a constant temperature of 35°C), due to the failure to perform cooling, experienced exponentially increased cellular energy consumption from prolonged high-temperature stress. This relentless overdraft is directly reflected in the sharp drop in ATP reserves shown in Table 2, which decreased to 6.18 μmol / g DCW at the 8-hour mark.

[0092] Figure 2In this system, the dissolved oxygen saturation curve showed an abnormal, continuous, and passive increase in the middle and later stages. This is essentially a macroscopic manifestation of the engineered strain experiencing large-scale death and loss of its own oxygen-consuming capacity after encountering energy depletion. Comparative Example 5 (one-time feeding), which changed the material addition sequence, went to the other extreme. The instantaneous loading of a massive amount of substrate directly broke through the processing limit of the tricarboxylic acid cycle, triggering… Figure 2 The system experienced a catastrophic oxygen crisis when the dissolved oxygen curve bottomed out, forcing carbon metabolism to shift towards a fermentation pathway involving substrate-level phosphorylation.

[0093] This behavior ultimately led to the accumulation of acetic acid at a level as high as 9.78 g / L. This deadly weak acidic toxin penetrated the cell membrane, destroyed the transmembrane proton gradient, caused ATP synthase to stop, and directly triggered... Figure 2 An abnormal rebound occurred when the dissolved oxygen saturation at the tail end approached 100%. The spatiotemporal metabolic decoupling operation successfully overcame the dual traps of energy depletion and metabolic toxicity by redefining the boundaries of biochemical reactions.

[0094] Test Example 3: This test case was used to determine the cumulative concentration of the final target product proline amide in each reaction system, thereby evaluating the effectiveness of different catalytic control strategies for whole-cell ammonolysis. The specific experimental steps are as follows: The reaction endpoint materials of Examples 1 to 4 and Comparative Examples 1 to 5 were selected as test objects. Samples were taken from each reactor and deionized water was added to restore homogeneity. The bacterial precipitate was removed by centrifugation at 10,000 rpm, and the supernatant containing the target product was collected and filtered through a 0.22 μm microporous membrane.

[0095] The filtered supernatant was pretreated using 2,4-dinitrofluorobenzene pre-column derivatization. The pretreated sample was then placed in an autosampler vial and analyzed by a Shimadzu high-performance liquid chromatography (HPLC) system.

[0096] The liquid chromatography system was configured with the following parameters: an amino acid analysis-specific liquid chromatography column (AAA 2.7 μm, 4.6 × 100 mm); a mobile phase of 50% (v / v) acetonitrile and 50 mmol / L sodium acetate mixture; a UV detector wavelength of 360 nm; a single injection volume of 10 μL; a mobile phase flow rate of 1 mL / min; and a constant column oven temperature of 40 °C. Chromatograms of each sample were recorded, and the target peak area within the retention time window was extracted. The concentration was calculated using the external standard method.

[0097] The quantitative test results are detailed in the table below: Table 3. HPLC quantitative analysis data of proline amide, the final product of the examples and comparative examples.

[0098] Conclusion Analysis: Combined with Table 3 and Figure 3 It can be seen that, Figure 3 This visually presents the signal distribution patterns of typical samples within a specific detection window under 360nm wavelength detection conditions. (Observation) Figure 3 The graph shows retention time (min) on the horizontal axis and ultraviolet absorption intensity (mAU) on the vertical axis. It can be seen that at the characteristic dashed line marked with a retention time of ≈7.0 min, the target substances in each system all exhibit elution absorption peaks, but their final accumulation amounts show macroscopic differences of orders of magnitude.

[0099] In Example 1, the chromatographic peak projection area dominated the space, with its UV absorption intensity exceeding 450 mAU, corresponding to a final proline amide concentration of 9.05 g / L in the fermentation broth in Table 3, and a substrate conversion rate exceeding 90%. For a long time, directly utilizing whole cells in a conventional aqueous environment to catalyze ammonolysis has faced significant yield bottlenecks. The test results of Comparative Example 1 (conventional buffer standard) objectively reflect this industry status quo.

[0100] Under the traditional static one-pot incubation mode, the chromatographic detection signal of this reference system is in Figure 3 The substrate almost perfectly adhered to the baseline of the instrument, resulting in a final conversion rate hovering around 1% and a product concentration of only 0.10 g / L. The irreversible hydrolytic destruction of the high-energy amidation intermediate by free water molecules constitutes the core obstacle preventing the substrate conversion chain from extending downstream in conventional aqueous systems.

[0101] Remodeling the cellular microenvironment plays a substantial role in blocking this hydrolytic competitive pathway. Figure 3 Comparative Example 3 (without PEG shielding) exhibited a moderate intensity chromatographic absorption peak profile. The data in Table 3 confirm that it is similar to Comparative Example 2, which lacks the salting-out trigger component, with the final product concentration hovering between 2.41 and 3.16 g / L.

[0102] Although both test systems employed low temperature and restricted feed-through operation, the lack of a polymer network or osmotic pressure shock prevented the successful construction of a microemulsion aggregation barrier around the cell membrane. Without the protection of the hydrophobic layer, the prolyl-AMP intermediate, with its extremely short half-life, was inevitably exposed again to the bulk aqueous solution. Hydrolysis side reactions regained control over substrate consumption, resulting in a persistently low conversion rate of around 30%.

[0103] The effective operation of whole-cell catalysis relies on a dual logical loop of external protection and continuous internal energy output. Even though Comparative Examples 4 and 5 completely preserved the phase transition triggering components and established a microemulsion isolation barrier outside the cells, their product yields showed a significant decline, accumulating only 1.06 g / L and 0.55 g / L, respectively. Isolation alone is insufficient to maintain the long-term operation of the entire catalytic engine. After decoupling the two key operations of temperature control and steady-state feeding, the overload of the carbon source and the severe depletion of cell maintenance energy due to continuous high temperatures caused the engineered strain to fall into a metabolic predicament of adenosine triphosphate depletion in the early stages of catalysis.

[0104] The disruption of intracellular energy supply directly cuts off the driving force for carboxylate reductase to activate substrates. The deep synergy between phase change microenvironment intervention and spatiotemporal metabolic decoupling in the process execution dimension fills the technical gaps in the material retention and energy regeneration stages of aqueous whole-cell preparation of proline amide.

Claims

1. A method for preparing proline catalyzed by carboxylic acid reductase, characterized in that, Includes the following steps: Pure water, basic buffer salt, polyethylene glycol 400, initial carbon source and recombinant whole-cell biocatalyst were added to the reactor and mixed evenly to form a homogeneous activation system. At a first temperature, the activation system is aerated and stirred; Turn on the cooling system. When the activation system cools down to the second temperature, inject a phase change trigger into the reactor. The sudden increase in salt concentration triggers mesoscopic phase separation, causing the activation system to transform into a microemulsion reaction system. The reaction system is further cooled and kept constant at a third temperature. Dual-channel feeding is started, wherein channel A continuously adds carbon source and dynamically adjusts to maintain the residual carbon source concentration in the reactor below a set threshold, while channel B continuously and uniformly adds a mixed aqueous solution of L-proline and ammonium salt. After the reaction system has completed its reaction, collect the product liquid containing proline amide.

2. The method for preparing proline catalyzed by carboxylic acid reductase according to claim 1, characterized in that, The polyethylene glycol 400 accounts for 4 wt% to 10 wt% of the total mass of the activation system; The initial carbon source is glucose, and the initial concentration of the initial carbon source in the activation system is 1.0 g / L to 3.0 g / L; The recombinant whole-cell biocatalyst reached an OD concentration in the activation system. 600 It ranges from 15 to 25.

3. The method for preparing proline catalyzed by carboxylic acid reductase according to claim 1, characterized in that, The preparation method of the recombinant whole-cell biocatalyst is as follows: Recombinant expression plasmids containing carboxyl reductase genes from Mycobacterium marineum and phosphoadenosyltransferase genes from Bacillus subtilis were transformed into competent Escherichia coli cells to obtain positive recombinant engineered strains. The positive recombinant engineered strain was inoculated into a fermentation medium, and isopropyl-β-D-thiogalactoside was added at a final concentration of 0.05 mmol / L to 0.2 mmol / L to induce culture for 16 to 24 h at a temperature of 16℃ to 30℃ to obtain the fermentation broth. The wet bacterial cells precipitated in the fermentation broth are collected by centrifugation to obtain the recombinant whole-cell biocatalyst.

4. The method for preparing proline catalyzed by carboxylic acid reductase according to claim 1, characterized in that, The specific composition and proportion of the basic buffer salt in the activation system are limited as follows: The concentration of MgSO4 was 0.1 g / L to 0.5 g / L; The concentration of Na2HPO4·12H2O is 10.0 g / L to 20.0 g / L; The concentration of KH2PO4 is 1.0 g / L to 5.0 g / L; The concentration of NaCl is 0.1 g / L to 1.0 g / L.

5. The method for preparing proline catalyzed by carboxylic acid reductase according to claim 1, characterized in that, The first temperature is 32℃~37℃, the stirring speed is 150rpm~250rpm, and the operation is maintained for 1.0h~2.0h.

6. The method for preparing proline catalyzed by carboxylic acid reductase according to claim 1, characterized in that, The second temperature is 28℃~29℃; The phase change trigger is anhydrous sodium sulfate. The phase change trigger is pumped into the reaction vessel instantaneously within 3 min to 10 min using a high-pressure metering pump, so that the final concentration of the phase change trigger in the reaction system rapidly reaches 30.0 g / L to 60.0 g / L.

7. The method for preparing proline catalyzed by carboxylic acid reductase according to claim 1, characterized in that, The third temperature is 22℃~26℃; In channel B, the total added amount of L-proline and the ammonium salt, converted to their respective concentrations in the reaction system, are as follows: The L-proline concentration is 8.0 g / L to 15.0 g / L, and the ammonium salt is ammonium chloride with a concentration of 4.0 g / L to 8.0 g / L. The feed time in channel B lasts from 8 to 14 hours.

8. The method for preparing proline catalyzed by carboxylic acid reductase according to claim 1, characterized in that, During the dual-channel feeding process, channel A is dynamically adjusted according to the carbon source consumption rate; The set threshold is 0.5 g / L to 1.0 g / L.

9. The method for preparing proline catalyzed by carboxylic acid reductase according to claim 1, characterized in that, The steps for collecting the product liquid containing proline amide include: adding 1 to 2 times the volume of deionized water to the reaction system after the reaction is completed to dilute and demulsify to restore homogeneity, then centrifuging to remove the bacterial cells, and collecting the supernatant to obtain the product liquid containing proline amide.

10. The method for preparing proline catalyzed by carboxylic acid reductase according to claim 3, characterized in that, The competent Escherichia coli cells were Escherichia coli BL21(DE3). The recombinant expression plasmid is pETDuet-1.

Images