Biological synthesis process of sialic acid and equipment thereof

Abstract

The invention relates to the technical field of sialic acid preparation, in particular to a biological synthesis process and equipment of sialic acid. The method comprises the following steps: carrying out fermentation culture on genetically modified engineering bacteria capable of efficiently expressing sialic acid synthetase in cells to obtain whole cells with high enzymatic activity; performing cell wall pretreatment and enzyme release treatment on the whole cells to obtain structural fatigue enzyme liquid; mixing the enzyme liquid with an immobilized carrier, and carrying out an immobilization reaction to obtain a solid immobilized enzyme; taking water as a solvent, synthesizing sialic acid by using the immobilized enzyme catalytic substrate, and recovering the immobilized enzyme through solid-liquid separation after the reaction; and purifying the reaction liquid from which the immobilized enzyme is separated to obtain a high-purity sialic acid product. According to the synthesis process disclosed by the invention, the enzyme activity yield reaches 86-90%, the immobilization efficiency is improved to 90-94%, the enzyme consumption cost can be greatly reduced, and high-purity sialic acid can be stably produced.

Description

Technical Field

[0001] This invention relates to the field of sialic acid preparation technology, and more specifically, to a biological synthesis process and equipment for sialic acid. Background Technology

[0002] Sialic acid (N-acetylneuraminic acid), a key component of cell membrane glycocomplexes, plays important physiological functions in neural development, antiviral mechanisms, and immune regulation. With its expanding applications, industrial production needs to meet the core requirements of non-toxic residues, high purity, and controllable costs.

[0003] Among various production methods, bio-enzymatic catalysis exhibits unique advantages due to its mild reaction conditions, high selectivity, and environmental friendliness. This method typically employs the following process route: first, sialic acid synthase is obtained through fermentation using genetically engineered bacteria; then, the enzyme is extracted from the bacterial cells and used to catalyze the conversion of the substrates N-acetylmmannosamine and pyruvate into sialic acid in a suitable aqueous reaction system. This process avoids the toxic catalysts used in chemical synthesis methods and overcomes the limitations of raw materials in natural extraction methods, offering significant advantages in terms of safety.

[0004] However, this technical route faces a key bottleneck in industrialization: the non-recoverable nature of the free enzyme catalyst leads to prohibitively high enzyme consumption costs. This problem stems from the inherent characteristics of the process: during the enzyme catalytic reaction stage, the free sialic acid synthase is directly dispersed in the aqueous reaction system, forming a homogeneous reaction system with the substrate and product. After the reaction, the enzyme molecules, the product sialic acid, unreacted substrate, and water form a stable homogeneous mixture. Because the enzyme protein and the target product sialic acid are highly similar in molecular weight range (both in the hundreds of Daltons), charge distribution characteristics, and water solubility, it is difficult to effectively separate and recover the enzyme from the reaction system using conventional physical separation methods such as centrifugation, filtration, and precipitation. Summary of the Invention

[0005] The purpose of this invention is to provide a biological synthesis process and equipment for sialic acid, so as to solve the problems of non-recoverable catalysts and high enzyme consumption costs caused by the use of free enzymes in existing biological enzyme catalysis methods.

[0006] To achieve the above objectives, one objective of this invention is to provide a biosynthetic process for sialic acid, comprising the following steps: S1. Fermentation culture of genetically modified engineered bacteria capable of efficiently expressing sialic acid synthase intracellularly to obtain whole cells with high enzyme activity; S2. Pre-treat the cell wall and release enzymes from the whole cells to obtain an enzyme solution for structural fatigue. S3. Mix the enzyme solution with the immobilization carrier and carry out the immobilization reaction to obtain a solid immobilized enzyme; S4. Using water as a solvent, immobilized enzymes are used to catalyze the synthesis of sialic acid from the substrate. The immobilized enzymes are then recovered through solid-liquid separation after the reaction. S5. The reaction solution after separating the immobilized enzyme is purified to obtain a high-purity sialic acid product. The structural fatigue refers to the micro-damage state formed after the bacterial cell wall is sheared by the cell wall pretreatment equipment. Its characteristics are that the cell wall integrity is damaged but the cell morphology is basically maintained, which facilitates the selective release of sialic acid synthase through mild physicochemical methods.

[0007] Based on this, step S2 includes: S2.1. The bacterial solution is introduced into the cell wall pretreatment equipment. The blunt protrusions on the surface of the rotor form a shear field with the stator, which applies a controllable mechanical force to the bacterial cells, causing micro-damage to the cell walls of the cells. S2.2. The pretreated bacterial solution is subjected to solid-liquid separation. The resulting bacterial cells are subjected to osmotic shock treatment in a low-temperature buffer to selectively release sialic acid synthase from the micro-damaged cells.

[0008] In the above technical solution, the core design of step S2 lies in achieving the goal of efficiently obtaining highly active enzyme solution from bacterial cells through the organic combination of physical and biochemical methods. Step S2.1 employs a cell wall pretreatment device with a specific structure. The blunt protrusions on the rotor surface and the stator form a controllable shear field, applying appropriate mechanical force to the bacterial cells, causing micro-damage to the cell wall and creating a structural fatigue state where the cell morphology is basically maintained but permeability is increased, thus creating the necessary conditions for subsequent enzyme release. Step S2.2, based on the pretreatment, subjectes the isolated bacterial cells to osmotic pressure shock treatment in a low-temperature buffer solution. Utilizing the sensitivity of micro-damaged cells to changes in osmotic pressure, the target enzyme with a smaller molecular weight preferentially permeates through the cell wall and is released into the liquid phase, while most high molecular weight impurities and cell debris are confined inside the cells. This avoids enzyme activity loss and impurity release caused by traditional violent disruption methods, improves the specific activity and purity of the enzyme solution, and provides high-quality enzyme raw materials for subsequent immobilization processes.

[0009] The second objective of this invention is to provide a biosynthetic apparatus for sialic acid, used in the biosynthetic process of sialic acid as described in any one of the above claims, comprising the following equipment arranged according to the process flow: Fermenters are used to cultivate engineered bacteria to obtain whole cells with high enzyme activity. A cell wall pretreatment device for performing cell wall shearing on the whole cells; A solid-liquid separation device is used to separate the solid and liquid components of a pretreated bacterial solution. An enzyme release reactor is used to subject the isolated bacterial cells to osmotic pressure shock treatment in order to obtain an enzyme solution. An immobilized enzyme preparation unit is used to mix the enzyme solution with an immobilized carrier to prepare an immobilized enzyme. An enzyme-catalyzed reactor for using the immobilized enzyme to catalyze the synthesis of sialic acid from a substrate in an aqueous phase; The product purification unit is used to purify the liquid after the catalytic reaction to obtain sialic acid product.

[0010] Based on this, the cell wall pretreatment equipment includes: The processing chamber contains a stator and a rotor, and the working surfaces of the rotor and the stator are provided with an array of non-sharp, blunt protrusions. The rotor is provided with a gap adjustment mechanism for adjusting the working gap between the rotor and the stator; A control system for controlling the gap adjustment mechanism and the motor that drives the rotor.

[0011] In this technical solution, the processing chamber in the cell wall pretreatment equipment provides a sealed and controllable working environment for cell processing. The blunt protrusion array on the rotor working surface works in conjunction with the blunt protrusion array on the stator working surface to form a shear field capable of applying appropriate mechanical force to the cells. This is the key structural basis for achieving micro-damage to the cell wall rather than complete fragmentation. The gap adjustment mechanism allows for precise adjustment of the working gap between the rotor and stator according to the characteristics of different cells, thereby controlling the intensity of the mechanical action and ensuring that the cells reach a specific state of structural fatigue. The control system is responsible for coordinating the operation of the drive motor and the gap adjustment mechanism, ensuring the consistency and reproducibility of the pretreatment effect by controlling core parameters such as rotational speed and gap.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. In the biosynthesis process and equipment for sialic acid, a cell wall pretreatment device with an array of non-sharp, blunt protrusions on the rotor and stator surfaces is used. This applies controllable mechanical shearing force to the whole-cell bacterial cells, causing micro-damage to the cell walls and achieving a state of structural fatigue. The cell morphology remains largely intact, but permeability is increased. Based on this, the treated cells are placed in a low-temperature buffer solution and subjected to osmotic pressure shock treatment. This selectively releases the smaller molecular weight sialic acid synthase from the micro-damaged cells, while most high molecular weight proteins and cell debris are confined intracellularly. This avoids the problems of enzyme conformational disruption and indiscriminate release of intracellular substances caused by traditional high-pressure homogenization, thereby increasing enzyme activity to 86% to 90% and reducing the release of proteins by approximately 40%, thus improving the specific activity and purity of the enzyme solution.

[0013] In this biosynthesis process and equipment for sialic acid, the obtained enzyme solution is immobilized with a porous polymer carrier under suitable conditions to form a solid immobilized enzyme. The immobilized enzyme can efficiently catalyze substrate conversion in a water-based catalytic system. After the reaction, it can be recovered and reused multiple times through simple solid-liquid separation, resulting in an immobilization efficiency of 90% to 94% and a significant improvement in enzyme utilization. This overcomes the bottlenecks of non-recoverable catalysts and high enzyme consumption costs in traditional free enzyme processes. Attached Figure Description

[0014] Figure 1 This is a flowchart of the synthesis process of the present invention; Figure 2 This is a flowchart of step 2 of the synthesis process of the present invention; Figure 3 This is a connection diagram of the device system of the present invention; Figure 4 This is a structural diagram of the cell wall pretreatment equipment of the present invention; Figure 5 For the present invention Figure 4 A schematic diagram of the structure of part A; Figure 6 This is a comparison diagram of cell states in this invention.

[0015] The meanings of the labels in the diagram are as follows: 1. Fermenter; 2. Cell wall pretreatment equipment; 21. Processing chamber; 22. Stator; 23. Rotor; 24. Array of blunt protrusions; 25. Gap adjustment mechanism; 26. Feed inlet; 27. Discharge outlet; 3. Solid-liquid separation device; 4. Enzyme release reactor; 5. Immobilized enzyme preparation unit; 6. Enzyme catalytic reactor; 7. Product purification unit. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0017] Example 1 Please see Figures 1-2 As shown, one of the objectives of this embodiment is to provide a biosynthetic process for sialic acid, including the following steps: S1. Fermentation culture of genetically modified engineered bacteria capable of efficiently expressing sialic acid synthase intracellularly to obtain whole cells with high enzyme activity; S2. Pre-treat the cell wall and release enzymes from the whole cells to obtain an enzyme solution for structural fatigue. Step S2 also includes: S2.1. The bacterial solution is introduced into the cell wall pretreatment device 2. The blunt protrusions on the surface of the rotor 23 and the stator 22 form a shear field, which applies a controllable mechanical force to the bacterial cells, causing micro-damage to the cell walls of the bacterial cells. S2.2. The pretreated bacterial solution is subjected to solid-liquid separation. The resulting bacterial cells are subjected to osmotic shock treatment in a low-temperature buffer to selectively release sialic acid synthase from the micro-damaged cells.

[0018] S3. Mix the enzyme solution with the immobilization carrier and carry out the immobilization reaction to obtain a solid immobilized enzyme; S4. Using water as a solvent, immobilized enzymes are used to catalyze the synthesis of sialic acid from the substrate. The immobilized enzymes are then recovered through solid-liquid separation after the reaction. S5. The reaction solution after separating the immobilized enzyme is purified to obtain a high-purity sialic acid product.

[0019] The structural fatigue refers to the micro-damage state formed after the bacterial cell wall is sheared by the cell wall pretreatment device 2. Its characteristics are that the cell wall integrity is damaged but the cell morphology is basically maintained, which facilitates the selective release of sialic acid synthase through mild physicochemical methods.

[0020] Therefore, the first step was to ferment and cultivate engineered bacteria. Genetically modified *E. coli* strains with the carnosine synthase gene were used, enabling them to autonomously synthesize ATP from adenine and methionine. These engineered bacteria were cultured in fermenter 1 in a medium containing carbon, nitrogen, and inorganic salts. High-density fermentation was carried out by controlling temperature and pH, and employing low-temperature induction and self-controlled feed-feed technology, ultimately yielding whole cells with high enzyme activity.

[0021] Subsequently, the whole cells underwent cell wall pretreatment and enzyme release treatment. The bacterial broth obtained from fermentation was passed into cell wall pretreatment equipment 2. Through the shear field formed between the hemispherical blunt protrusions on the surface of the rotor 23 and the stator 22, a controllable mechanical force was applied to the bacterial cells under precisely controlled working intervals and rotation speeds, causing micro-damage to the cell walls and achieving a state of "structural fatigue" where the cell morphology was basically maintained but the integrity was compromised. The pretreated bacterial broth was then subjected to solid-liquid separation, and the resulting bacterial cells were resuspended in a low-temperature buffer solution. Osmotic pressure shock treatment was used to selectively release sialic acid synthase from the micro-damaged cells, obtaining a clear enzyme solution.

[0022] The enzyme solution was mixed with a porous polymer immobilization carrier at a suitable temperature to carry out an immobilization reaction. After the reaction was completed, the immobilized enzyme was obtained by filtration and washing. Then, using water as a solvent, the immobilized enzyme and substrate solution were added to an enzyme catalytic reactor 6, and a catalytic synthesis reaction was carried out under controlled temperature and pH conditions. After the reaction was completed, the immobilized enzyme was recovered by solid-liquid separation, realizing the recycling of the catalyst.

[0023] Finally, the reaction solution after isolating the immobilized enzyme was purified. The reaction solution was first separated by ion exchange chromatography to collect the target sialic acid component; then the collected solution was concentrated by membrane concentration technology, and the concentrate was decolorized by adding activated carbon under inert gas protection. After decolorization, it was filtered, and the filtrate was transferred to a crystallization device for crystallization by programmed temperature control. The obtained crystals were centrifuged, washed, and dried under vacuum to finally obtain a high-purity sialic acid solid product.

[0024] Example 2 This embodiment provides a complete equipment system for the biosynthesis process of sialic acid. For example... Figure 3 As shown, the equipment system is configured according to the process flow of sialic acid biosynthesis, and includes, in sequence: a fermenter 1 for engineered bacteria cultivation, a cell wall pretreatment device 2 for treating cell wall micro-damage, a solid-liquid separation device 3 for cell separation, an enzyme release reactor 4 for enzyme release, an immobilized enzyme preparation unit 5 for catalyst preparation, an enzyme catalytic reactor 6 for the synthesis reaction, and a product purification unit 7 for product refining. These equipment units are sequentially connected via material conveying pipelines to form a continuous production line, realizing the integration of the entire process from strain cultivation to final product acquisition.

[0025] First, genetically modified engineered bacteria are fermented and cultured in fermenter 1 to obtain whole cells with high enzyme activity. Fermenter 1 is made of stainless steel, and its jacketed temperature control system maintains the temperature inside the tank within the suitable range for the growth of engineered bacteria through a circulating heat transfer medium. A mechanical agitator inside the tank continuously stirs the fermentation broth, ensuring uniform mixing of the bacteria and nutrients. A pH electrode monitors the acidity or alkalinity of the fermentation broth; when the detected value exceeds the set range, an automatic feed system adds acid or alkali solution to adjust the pH. A dissolved oxygen electrode monitors the dissolved oxygen concentration in the fermentation broth; when the concentration is insufficient, the system increases the dissolved oxygen by increasing the stirring speed or adjusting the air intake. Sterile air is supplied to fermenter 1 through a sterilization filter to meet the oxygen requirements for the growth of engineered bacteria. An exhaust gas treatment device condenses and filters the gases produced during fermentation, providing a stable and reliable culture environment for high-density fermentation and high enzyme activity expression of engineered bacteria, and providing qualified whole-cell raw materials for subsequent process steps.

[0026] like Figure 4 and Figure 5As shown, the cell wall pretreatment equipment 2 is used to perform controlled mechanical shearing treatment on whole cells obtained from fermentation, causing structural fatigue of the cell walls. The cell wall pretreatment equipment 2 mainly includes a treatment chamber 21, a stator 22, and a rotor 23. The treatment chamber 21 is a sealed container made of stainless steel, equipped with an inlet 26 and an outlet 27. It is externally fitted with an insulation jacket, which maintains a low-temperature environment inside the chamber through a circulating cooling medium to prevent damage to enzyme activity during treatment. The rotor 23 is mounted inside the treatment chamber 21 via a main shaft. Its working surface is precision-machined and features a regularly arranged array of hemispherical blunt protrusions 24. The stator 22 is fixed to the inner wall of the chamber and concentrically arranged with the rotor 23. Its inner surface has concave structures corresponding to the protrusions of the rotor 23. When the rotor 23 rotates at high speed, a high-frequency changing shear field is formed between the stator 22 and the rotor 23, exerting mechanical effects such as compression and stretching on the passing bacterial solution. The gap adjustment mechanism 25 is connected to the main shaft of the rotor 23 and uses a servo motor drive combined with a ball screw transmission method to finely adjust the working gap between the rotor 23 and the stator 22. The control system is electrically connected to the equipment motor and the gap adjustment mechanism 25. Based on the received bacterial parameter signals, it automatically calculates and sets the rotor speed and working gap size, and selects the optimal treatment conditions for different bacterial characteristics.

[0027] During operation, the bacterial solution is pumped into the processing chamber 21 through the inlet 26, where it is subjected to controlled mechanical forces within the shear field formed by the rotor 23 and stator 22. The bacterial cell walls undergo micro-damage under the repeated action of the blunt protrusions, reaching a state of structural fatigue where the cell morphology is largely maintained but permeability is increased. The treated bacterial solution is discharged from the outlet 27 and proceeds to the next process. The entire process is carried out at low temperatures, effectively protecting the enzyme's biological activity.

[0028] Solid-liquid separation device 3 is used to separate the bacterial solution after cell wall pretreatment to obtain bacterial cells for subsequent enzyme release treatment. This device employs high-speed centrifugal separation technology and mainly consists of a centrifugal drum, drive system, feeding and discharging devices, and control system. The centrifugal drum is precision-manufactured from stainless steel and features a special disc assembly structure. When the bacterial solution enters the high-speed rotating drum through the feeding device, the bacterial cells and liquid rapidly separate under centrifugal force. The bacterial cells deposit on the inner wall of the drum and are discharged through a specific slag discharge mechanism; the separated clear liquid is discharged through the overflow port at the top of the drum. The drive system provides stable rotational power to the equipment, ensuring the continuity of the separation process. The control system precisely adjusts the drum speed and feed rate to adapt to different processing volumes. This device is directly connected to the cell wall pretreatment device 2 to receive the bacterial solution after micro-damage treatment. The separation operation is completed under low-temperature conditions, effectively preventing the loss of enzyme activity. The separated bacterial cells are immediately transported to the enzyme release reactor 4 for subsequent processing, ensuring the continuity and efficiency of the entire process.

[0029] Enzyme release reactor 4 is used to subject the isolated bacterial cells to osmotic shock treatment, selectively releasing sialic acid synthase. This equipment mainly includes a reaction vessel, a temperature control system, a stirring device, and a pH monitoring unit. The reaction vessel has a jacketed structure, and the processing temperature is precisely controlled by circulating heat transfer medium. The stirring device is driven by a variable frequency motor, and the stirring intensity can be adjusted according to process requirements to ensure thorough mixing of the bacterial cells and buffer solution. The pH monitoring unit monitors the pH changes of the reaction system in real time, providing a basis for process control.

[0030] During operation, the bacterial cells from the solid-liquid separation device 3 are mixed with a specific concentration of cryogenic buffer in a reaction vessel. By controlling the temperature, stirring intensity, and processing time, changes in osmotic pressure are used to selectively release sialic acid synthase from cells with micro-damage. After processing, the resulting enzyme solution is discharged through the outlet and enters the subsequent immobilized enzyme preparation process.

[0031] Immobilized enzyme preparation unit 5 is used to convert enzyme solution into reusable solid immobilized enzyme. This unit includes a mixing reaction vessel, a temperature control system, and a filtration and collection system. The mixing reaction vessel is equipped with a speed-regulating stirrer to ensure sufficient contact between the enzyme solution and the porous polymer carrier; the temperature control system maintains the required temperature for the immobilization reaction through a jacketed circulating water bath; the filtration and collection system achieves solid-liquid separation through a filter membrane with a specific pore size. During the preparation process, the enzyme solution and the carrier undergo an immobilization reaction under optimized conditions. After the reaction is completed, the immobilized enzyme is obtained by vacuum filtration, and then washed with buffer to remove unimmobilized enzyme molecules. The prepared immobilized enzyme is directly transferred to the enzyme catalytic reactor 6 through a closed delivery system.

[0032] Enzyme-catalyzed reactor 6 is used for the catalytic synthesis of sialic acid from substrates in an aqueous environment. This reactor is a stirred tank type, equipped with a temperature control system, an automatic pH adjustment device, and a solid-liquid separation interface. During the reaction, the immobilized enzyme and substrate solution undergo catalytic reaction under strictly controlled temperature and pH conditions. The automatic pH adjustment system maintains the stability of the reaction system through online monitoring and automatic addition of acid and alkali solutions. After the reaction, a special filtration structure at the bottom of the reactor enables rapid separation of the immobilized enzyme from the reaction solution. The separated immobilized enzyme, after washing with buffer solution, can be directly used in the next batch of reaction, achieving efficient recycling of the catalyst.

[0033] Product purification unit 7 is used to purify the catalytic reaction solution to obtain high-purity sialic acid. This unit is configured sequentially according to the process flow, including an ion exchange chromatography system, a membrane concentration unit, a decolorization tank, a crystallization vessel, and a vacuum dryer. The ion exchange chromatography system selectively adsorbs sialic acid using a specific type of anion exchange resin, followed by gradient elution to separate and purify the target product. The membrane concentration unit employs nanofiltration membrane technology to gently concentrate the purified solution under suitable pressure. The decolorization tank removes pigment impurities through activated carbon adsorption under controlled temperature conditions. The crystallization vessel achieves controlled crystallization of sialic acid through a programmed cooling control system. Finally, the crystallized product is centrifuged and then dried at low temperature in a vacuum dryer to obtain high-purity sialic acid that meets pharmaceutical requirements.

[0034] Comparative Example This comparative example details the technical differences between the traditional high-pressure homogenization process for free enzyme catalysis and the process of this invention.

[0035] Traditional processes employ high-pressure homogenization to completely disrupt bacterial cells, thoroughly destroying the cell wall structure through high-intensity mechanical force. Cell debris is then removed via high-speed centrifugation to obtain a crude enzyme solution. This process is violent, easily leading to enzyme protein denaturation and inactivation. Furthermore, cell debris removal is difficult, and residual impurities interfere with subsequent purification processes.

[0036] like Figure 6 As shown, the process of this invention employs an innovative cell wall pretreatment technology. A controllable shear field is formed between the blunt protrusions on the surface of rotor 23 and stator 22, causing micro-damage to the bacterial cell wall rather than complete breakage, resulting in a state of structural fatigue. Subsequently, a gentle osmotic pressure shock treatment selectively releases intracellular enzymes, maximizing the preservation of the enzyme's native conformation and biological activity.

[0037] Comparison parameters Traditional process (high-pressure homogenous crushing) The process of this invention (controllable pretreatment of cell walls) Core Operations Under high pressure, the bacterial solution is forced through narrow gaps, and the cells are completely broken down by shearing and impact forces. The blunt protrusions of rotor 23 exert gentle, controlled mechanical shearing on cells within a specific gap. degree of cell fragmentation Completely broken, cell structure irreparable Micro-damage, cell morphology basically intact Target enzyme (sialic acid synthase) activity 68-72% 86-90% Release Selectivity All intracellular substances (including miscellaneous proteins, nucleic acids, etc.) are released indiscriminately. The target enzyme is released preferentially, reducing the release of other proteins by approximately 40%. Cell debris content in the enzyme solution after treatment High, requires multiple high-speed centrifugation removals Extremely low levels, no special cell debris removal steps required. Preserving the enzyme's native conformation to the greatest extent possible Poor quality; high pressure and high speed can easily lead to localized enzyme denaturation. Good, mild shear force effectively protects the enzyme's spatial structure. Impact on subsequent immobilization efficiency The enzyme solution contains many impurities, and the immobilization carrier is easily contaminated, resulting in an efficiency typically of 75-80%. The enzyme solution is pure, and the immobilization efficiency is increased to 90-94%. Based on the process effect comparison data shown in Table 1, the cell wall controllable pretreatment process is superior to the traditional high-pressure homogenization crushing process in many key indicators. The specific analysis is as follows: At the core operational level, traditional processes rely on high-intensity physical action to achieve complete cell disruption, while controllable cell wall pretreatment processes achieve gentle processing through mechanical shearing of specific structures. This fundamental difference directly determines the differentiation of subsequent technical indicators.

[0038] Regarding enzyme extraction efficiency, the controllable cell wall pretreatment process increased the target enzyme activity from 68-72% in traditional processes to 86-90%, while simultaneously achieving selective release of the target enzyme and reducing the release of impurity proteins by approximately 40%. This indicates that the process not only improves enzyme extraction efficiency but, more importantly, enhances enzyme quality. In subsequent processing, the controllable cell wall pretreatment process exhibits even more significant advantages. The cell debris content in the enzyme solution after treatment is extremely low, eliminating the need for a dedicated removal step; simultaneously, it better preserves the enzyme's native conformation, creating favorable conditions for the subsequent immobilization process. This increases the immobilization efficiency from 75-80% in traditional processes to 90-94%.

[0039] In summary, the controllable cell wall pretreatment process achieves efficient and highly selective extraction of target enzymes by maintaining the basic integrity of cell morphology through a gentle and controllable action. This not only improves enzyme activity and purity but also lays a solid foundation for the optimization of subsequent process steps, forming a systematic technical advantage.

[0040] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for the biosynthetic synthesis of sialic acid, characterized in that, The methods and steps include the following: S1. Fermentation culture of genetically modified engineered bacteria capable of efficiently expressing sialic acid synthase intracellularly to obtain whole cells with high enzyme activity; S2. Pre-treat the cell wall and release enzymes from the whole cells to obtain an enzyme solution for structural fatigue. S3. Mix the enzyme solution with the immobilization carrier and carry out the immobilization reaction to obtain a solid immobilized enzyme; S4. Using water as a solvent, immobilized enzymes are used to catalyze the synthesis of sialic acid from the substrate. The immobilized enzymes are then recovered through solid-liquid separation after the reaction. S5. The reaction solution after separating the immobilized enzyme is purified to obtain a high-purity sialic acid product. The structural fatigue refers to the micro-damage state formed after the bacterial cell wall is sheared by the cell wall pretreatment device (2). Its characteristics are that the cell wall integrity is damaged but the cell morphology is basically maintained, which facilitates the selective release of sialic acid synthase through mild physicochemical methods.

2. The biosynthetic process for sialic acid according to claim 1, characterized in that, Step S2 includes: S2.

1. The bacterial solution is introduced into the cell wall pretreatment device (2). The blunt protrusions on the surface of the rotor (23) and the stator (22) form a shear field, which applies a controllable mechanical force to the bacterial cells, causing micro-damage to the cell walls of the bacterial cells. S2.

2. The pretreated bacterial solution is subjected to solid-liquid separation. The resulting bacterial cells are subjected to osmotic shock treatment in a low-temperature buffer to selectively release sialic acid synthase from the micro-damaged cells.

3. The biosynthetic process for sialic acid according to claim 2, characterized in that: The blunt protrusion has a hemispherical structure.

4. The biosynthesis process of sialic acid according to claim 1, characterized in that: The engineered bacteria were constructed by introducing a carnosine synthase gene into the host bacteria, enabling them to autonomously synthesize ATP using adenine and methionine.

5. The biosynthesis process of sialic acid according to claim 4, characterized in that: The engineered bacteria were fermented under low-temperature induction and self-controlled feedback conditions.

6. The biosynthesis process of sialic acid according to claim 1, characterized in that: The purification process includes sequentially performing ion exchange separation and membrane concentration on the reaction solution.

7. The biosynthesis process of sialic acid according to claim 1, characterized in that: The immobilization carrier is a porous polymer carrier.

8. A biosynthetic apparatus for sialic acid, used in the biosynthetic process comprising any one of claims 1-7, characterized in that, This includes the following equipment set up according to the process flow: Fermenter (1) is used to cultivate engineered bacteria to obtain whole cells with high enzyme activity; Cell wall pretreatment equipment (2) is used to perform cell wall shearing treatment on the whole cells; Solid-liquid separation device (3) is used to separate the pretreated bacterial solution from the solid solution; Enzyme release reactor (4) is used to subject the isolated bacterial cells to osmotic pressure shock treatment in order to obtain enzyme solution; The immobilized enzyme preparation unit (5) is used to mix the enzyme solution with the immobilized carrier to prepare the immobilized enzyme; An enzyme catalytic reactor (6) is used to catalyze the synthesis of sialic acid from a substrate in an aqueous phase using the immobilized enzyme; The product purification unit (7) is used to purify the liquid after the catalytic reaction to obtain sialic acid product.

9. The biosynthesis apparatus for sialic acid according to claim 8, characterized in that: The cell wall pretreatment device (2) includes: The processing chamber (21) is provided with a stator (22) and a rotor (23). The working surfaces of the rotor (23) and the stator (22) are provided with an array of non-sharp, blunt protrusions (24). The rotor (23) is provided with a gap adjustment mechanism (25) for adjusting the working gap between the rotor (23) and the stator (22); A control system for controlling the gap adjustment mechanism (25) and the motor that drives the rotor (23).

10. The biosynthesis apparatus for sialic acid according to claim 9, characterized in that: The control system is communicatively connected to an online detection unit used to monitor the physiological parameters of the fermentation broth cells.

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