A process for the production of enzymes by enzymatic hydrolysis

Abstract

The application provides a kind of for complex enzyme preparation with enzymatic hydrolysis process, it is related to enzyme purification preparation technical field, including the following steps: strain breeding and genetic engineering optimization;Using fed-batch fermentation technology carries out high-density fermentation culture;Multi-stage separation and purification, including centrifugal treatment and ion chromatography process;Material stirring mixing;Using jacketed stirring reaction tank carries out continuous enzymolysis, phase change material in jacket layer absorbs reaction heat, and temperature is controlled by semiconductor temperature control module;Flash sterilization treatment, let enzyme lose activity, prevent excessive decomposition, the application can also directly transport solution to the ion chromatography component in bottom after centrifugation and carry out subsequent chromatography process, reduce the equipment cost, also reduce the probability of solution being contaminated, can also increase the power output of driving mechanism after completing chromatography, realize the scraping effect of chromatography channel inner wall, improve the output rate, improve the yield of enzyme.

Description

Technical Field

[0001] This invention relates to the field of enzyme purification and preparation technology, specifically to an enzymatic hydrolysis process for the preparation of compound enzymes. Background Technology

[0002] Complex enzymes are multi-component enzyme systems composed of various complementary enzyme preparations (such as proteases, amylases, and cellulases) combined in specific proportions. Through synergistic effects, they can efficiently decompose multi-component structures in complex substrates, significantly improving enzymatic hydrolysis efficiency and product yield. Compared to single enzymes, complex enzymes have broader substrate adaptability and can specifically break down anti-nutritional factors or macromolecular cross-linked networks in raw materials. They are widely used in food processing (such as the hydrolysis of animal and plant proteins), bioenergy production, and pharmaceutical intermediate production. Modern processes often design customized complex formulations based on the characteristics of raw materials to achieve precise enzymatic hydrolysis and cost optimization.

[0003] In existing enzymatic hydrolysis processes, the preparation of complex enzymes involves multiple purification steps, including centrifugation, filtration, and chromatography. As a result, the equipment is expensive and inefficient. Frequent solution transfers are required, increasing the probability of contamination. Furthermore, the enzymes tend to adhere to the inner wall during chromatography, making them difficult to remove and reducing the final yield. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an enzymatic hydrolysis process for the preparation of compound enzymes, thereby solving the problems mentioned in the background art. The present invention also enables the solution to be directly transported to the bottom ion chromatography assembly after centrifugation for subsequent chromatography processes, reducing equipment costs and the probability of solution contamination. Furthermore, it increases the power output of the drive mechanism after chromatography, achieving a scraping effect on the inner wall of the chromatography channel, improving yield and enzyme production. The combined use of ion exchange chromatography and affinity chromatography achieves a purity of over 95%, while preserving the enzyme's active conformation.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: an enzymatic hydrolysis process for preparing compound enzymes, comprising the following steps:

[0006] S1. Strain selection and genetic engineering optimization to modify enzyme-producing strains and enhance the expression of the target enzyme encoding gene;

[0007] S2. High-density fermentation culture is carried out using fed-batch fermentation technology;

[0008] S3. Multi-stage separation and purification, including centrifugation and ion chromatography. The solution inside the centrifuge tank is concentrated to the side by centrifugation and pressure is generated. Finally, the enzyme in the solution is transported to the bottom ion chromatography module through the filter membrane for chromatographic purification. Selective adsorption and separation are achieved by electrostatic interaction between the chromatographic channels with opposite charges and the target enzyme or protein.

[0009] S4. Material mixing: After the pre-treated crushed raw material is fed into the mixing chamber through a screw conveyor belt, the material is subjected to a triple action of shearing, impact and cavitation effect under high pressure.

[0010] S5. Continuous enzymatic hydrolysis is carried out using a jacketed stirred reaction vessel. The phase change material in the jacket layer absorbs the heat of reaction, and the temperature is controlled by a semiconductor temperature control module.

[0011] S6. Flash sterilization treatment deactivates enzymes and prevents excessive decomposition.

[0012] Furthermore, in step S1, by integrating gene editing and directed evolution technologies, the enzyme-producing strain is precisely modified to target and enhance the expression of the encoding gene of the target enzyme, and a high-throughput screening platform is used to screen out highly efficient mutant strains that are resistant to extreme environments.

[0013] Furthermore, in step S2, a real-time online sensor control system integrating dissolved oxygen, carbon-nitrogen ratio, and inducer concentration is integrated. At the same time, metal ions such as Ca²⁺ and Mg²⁺ are added as cofactors to activate the active center of enzyme protein folding. The accumulation of metabolic byproducts is inhibited through a glucose gradient-limited feeding strategy.

[0014] Furthermore, in step S3, the raw materials for enzyme preparation are injected into the centrifuge assembly through the injection pipe at the top, and the motor in the drive mechanism is started. The motor drives the drive shaft to rotate, and the top of the drive shaft drives the toothed belt to rotate through the second gear.

[0015] Furthermore, the toothed belt drives the injection pipe in the centrifuge assembly to rotate, thereby causing the bottom linkage shaft and push plate to rotate at high speed inside the centrifuge tank.

[0016] Furthermore, the enzyme solution enters the ion chromatography assembly and adsorbs onto the chromatography medium on the inner wall of the chromatography channel. During this process, the motor in the drive mechanism is activated, which drives the drive shaft to rotate. The drive shaft drives the surface drive gear to rotate, and the drive gear is embedded in the fixed track, driving the rotating ring inside the fixed track. With the help of the outer and inner gear rings, each fourth gear is controlled to rotate, so that each ion chromatography assembly can rotate.

[0017] Furthermore, the driven column drives the central shaft at the bottom to rotate. When the central shaft rotates, it pulls the scraper embedded inside to rotate through the guide plates at the top and bottom. The scraper pushes the flowing enzyme solution when the rotation speed is relatively slow, and the enzyme solution in the auxiliary center also comes into contact with the chromatography medium on the inner wall of the chromatography channel.

[0018] Furthermore, after completing the chromatographic purification, by increasing the speed of the motor, the rotation speed of the central shaft is increased, causing the scraper to generate greater centrifugal force. At this point, the magnetic column on the inner side of the scraper can be detached from the magnetic sleeves on both sides, allowing the scraper to press directly against the inner wall of the chromatography channel. In the subsequent rotation process, the enzyme solution adhering to the chromatography medium is directly scraped off, completing the enzyme purification process.

[0019] Furthermore, in S6, the liquid material after microbial inactivation pretreatment is accelerated by a high-pressure plunger pump through the synergistic effect of ultra-high temperature instantaneous treatment and vacuum flash cooling, and atomized into micron-sized droplets through a spiral microchannel nozzle, and heat exchanged with superheated steam during the spraying process.

[0020] Furthermore, the amount of steam is dynamically adjusted by detecting changes in the light transmittance of the material, and blockchain technology is used to record the sterilization temperature-time curve. Finally, the liquid is converted into a solid state through a spray drying tower.

[0021] The beneficial effects of this invention are:

[0022] 1. In the enzymatic hydrolysis process for preparing compound enzymes, a single drive mechanism controls the centrifugal assembly at the top to rotate at high speed, thereby centrifuging the internal solution portion. After centrifugation, the solution can be directly transported to the ion chromatography assembly at the bottom for subsequent chromatography, reducing equipment costs and the probability of solution contamination.

[0023] 2. The enzymatic hydrolysis process for preparing compound enzymes uses a drive mechanism to control the rotation of the central shaft inside the ion chromatography assembly. During the chromatography process, the ion chromatography assembly slowly agitates the solution inside, ensuring that the solution can be adsorbed onto the inner wall of the chromatography channel. At the same time, after the chromatography is completed, the power output of the drive mechanism can be increased to achieve a scraping effect on the inner wall of the chromatography channel, thereby improving the yield.

[0024] 3. This enzymatic hydrolysis process for preparing compound enzymes adds metal ions as cofactors, which improves enzyme synthesis efficiency, shortens the fermentation cycle, and increases enzyme yield. The combination of ion exchange chromatography and affinity chromatography achieves a purity of over 95% while preserving the enzyme's active conformation. Attached Figure Description

[0025] Figure 1 This is a flowchart of an enzymatic hydrolysis process for preparing compound enzymes according to the present invention;

[0026] Figure 2 This is a structural diagram of the purification tank section in the enzymatic hydrolysis process for preparing compound enzymes according to the present invention;

[0027] Figure 3 This is an internal cross-sectional view of the purification tank of the present invention;

[0028] Figure 4 This is a schematic diagram of the drive mechanism of the present invention;

[0029] Figure 5 This is an exploded view of the centrifuge component of the present invention;

[0030] Figure 6 for Figure 3 Enlarged view of region A in the middle;

[0031] Figure 7 This is a schematic diagram of the structure of the ion chromatography component of the present invention;

[0032] Figure 8 This is a top view of the interior of the fixed track of the present invention;

[0033] In the diagram: 1. Purification tank; 2. Base; 3. First extension plate; 4. Second extension plate; 5. Drive mechanism; 6. Centrifuge assembly; 7. Centrifuge tank; 8. Ion chromatography assembly; 9. Motor; 10. Drive shaft; 11. First gear; 12. Second gear; 13. Toothed belt; 14. Fixed track; 15. Fixed sleeve; 16. Guide pipe; 17. Movable collar; 18. Arc plate; 19. Filter membrane; 20. Injection pipe; 21. Third gear; 22. Linkage shaft; 23. Push plate; 24. Rotating ring; 25. External toothed ring; 26. Internal toothed ring; 27. Driven column; 28. Support frame; 29. ​​Chromatography channel; 30. Fourth gear; 31. Central shaft; 32. Guide plate; 33. Slide groove; 34. Scraper; 35. Magnetic column; 36. Magnetic sleeve. Detailed Implementation

[0034] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0035] Please see Figures 1 to 8 The present invention provides the following technical solution: an enzymatic hydrolysis process for preparing compound enzymes, comprising the following steps:

[0036] S1. Strain Selection and Genetic Engineering Optimization: Using CRISPR-Cas9 or directed evolution technology, enzyme-producing strains are modified to enhance the expression of the target enzyme's encoding gene. This step integrates CRISPR-Cas9 gene editing and directed evolution technology to precisely modify enzyme-producing strains, targeting and enhancing the expression of the target enzyme's encoding gene. High-throughput screening platforms are used to identify highly efficient mutant strains tolerant to extreme environments (high temperature 60-80℃, wide pH range 3-11). Combined with metabolomics analysis, the carbon source utilization pathway and cofactor supply of the strains are optimized, simultaneously improving enzyme synthesis rate and stability, laying a foundation for efficient biosynthesis in subsequent high-density fermentation.

[0037] S2, high-density fermentation culture, employs fed-batch fermentation technology. It utilizes a real-time online sensor control system integrating dissolved oxygen, carbon-nitrogen ratio, and inducer concentration, while adding metal ions such as Ca²⁺ and Mg²⁺ as cofactors to activate the active center of enzyme protein folding, significantly improving synthesis efficiency. A glucose gradient-limited feeding strategy is used to inhibit the accumulation of metabolic byproducts, providing highly active enzyme solution raw materials for subsequent large-scale separation and purification.

[0038] S3. Multi-stage separation and purification, including centrifugation and ion chromatography processes. This step uses purification tank 1. The top of purification tank 1 includes a drive mechanism 5 and a centrifugation assembly 6. The drive mechanism 5 includes a motor 9, a drive shaft 10, a first gear 11, a second gear 12, and a toothed belt 13. A first extension plate 3 is welded to the top side of purification tank 1, and a second extension plate 4 is welded to the side of centrifugation tank 7. The drive shaft 10 passes through the middle of the first extension plate 3 and the second extension plate 4 in sequence. The motor 9 is screwed to the bottom of the first extension plate 3.

[0039] The bottom of the centrifuge tank 7 is provided with an arc-shaped plate 18, and a flow guide pipe 16 is opened on the surface of the arc-shaped plate 18. A filter membrane 19 is laid on the top of the flow guide pipe 16. A movable collar 17 is fitted on the top of the centrifuge tank 7. An injection pipe 20 is inserted into the inner side of the movable collar 17. A third gear 21 is integrally formed on the surface of the injection pipe 20. A toothed belt 13 is fitted on the surface of the third gear 21. A linkage shaft 22 is connected to the bottom of the injection pipe 20. A push plate 23 is welded to the side of the linkage shaft 22. The bottom of the push plate 23 is used to press against the surface of the arc-shaped plate 18.

[0040] The raw materials for enzyme preparation are injected into the centrifuge assembly 6 through the injection pipe 20 at the top. The motor 9 in the drive mechanism 5 is started, and the motor 9 drives the drive shaft 10 to rotate. The top of the drive shaft 10 drives the toothed belt 13 to rotate through the second gear 12. The toothed belt 13 drives the injection pipe 20 in the centrifuge assembly 6 to rotate, thereby causing the bottom linkage shaft 22 and push plate 23 to rotate at high speed inside the centrifuge tank 7. This centrifugal effect causes the solution inside the centrifuge tank 7 to concentrate to the side and generate pressure. Finally, the enzyme in the solution is transported to the bottom ion chromatography assembly 8 through the filter membrane 19.

[0041] The ion chromatography assembly 8 includes a driven column 27, a central shaft 31, a guide plate 32, and a scraper. A chromatography channel 29 is provided inside the purification tank 1. A fixing sleeve 15 is welded to the top of the chromatography channel 29. The driven column 27 is embedded inside the fixing sleeve 15. A fourth gear 30 is provided on the side of the driven column 27. A support frame 28 is welded to the inner wall of the driven column 27. The central shaft 31 is installed at the bottom of the support frame 28. A guide plate 32 is integrally formed on the surface of the central shaft 31. A groove 33 is provided on the surface of the guide plate 32. A scraper is inserted inside the groove 33. A magnetic suction column 35 is attached to the side of the scraper. A magnetic suction shell 36 is installed on the surface of the guide plate 32. The magnetic suction shell 36 is used to attract the magnetic suction column 35. The first gear 11 is used to mesh with the fourth gear 30.

[0042] The enzyme solution enters the ion chromatography assembly 8 and adsorbs onto the chromatography medium on the inner wall of the chromatography channel 29. During this process, the motor 9 in the drive mechanism 5 is activated, which drives the drive shaft 10 to rotate. The drive shaft 10 drives the surface drive gear to rotate. The drive gear is embedded in the fixed track 14, which drives the rotating ring 24 inside the fixed track 14. The outer gear ring 25 and the inner gear ring 26 simultaneously control the rotation of each fourth gear 30, so that each ion chromatography assembly 8 can rotate.

[0043] The driven column 27 drives the central shaft 31 at the bottom to rotate. When the central shaft 31 rotates, it pulls the scraper 34 embedded in the inner side to rotate through the guide plates 32 at the top and bottom. The scraper 34 pushes the flowing enzyme solution when the rotation speed is relatively slow, and the enzyme solution in the auxiliary center also comes into contact with the chromatography medium on the inner wall of the chromatography channel 29.

[0044] After the chromatography purification is completed, the rotation speed of the central shaft 31 is increased by increasing the rotation speed of the motor 9, which causes the scraper 34 to generate a greater centrifugal force. At this time, the magnetic suction column 35 on the inner side of the scraper 34 can be separated from the magnetic suction sleeve 36 on both sides, so that the scraper 34 can be pressed directly against the inner wall of the chromatography channel 29. Then, in the subsequent rotation process, the enzyme solution adhering to the chromatography medium is directly scraped off, thus completing the purification process of the enzyme.

[0045] Selective adsorption and separation are achieved through electrostatic interactions between the chromatographic channel 29 with the target enzyme or protein, which carries an opposite charge. During the procedure, precise control of the buffer pH and ionic strength forces impurity proteins to elute preferentially due to charge shielding, while the target enzyme desorbs under specific ionic strength, thus improving purity.

[0046] S4. Material mixing: After the pre-treated crushed raw material is fed into the mixing chamber via a screw conveyor belt, it is subjected to a triple action of shearing, impact and cavitation under high pressure. At this time, the intelligent temperature control system circulates heat transfer oil through the jacket layer to stabilize the material temperature in the optimal range of enzymatic hydrolysis of 50±0.5℃. Finally, the homogeneous slurry that meets the requirements of enzymatic hydrolysis is output through the membrane filter device at the bottom conical discharge port.

[0047] S5. A jacketed stirred reaction vessel is used for continuous enzymatic hydrolysis. The phase change material in the jacket absorbs the heat of reaction, and the temperature is precisely stabilized at 55±0.1℃ by a semiconductor temperature control module. At the same time, the pH electrode array detects the acidity and alkalinity in real time and automatically injects citrate buffer to maintain the optimal enzyme activity window of pH 6.8.

[0048] S6. Flash sterilization deactivates enzymes and prevents excessive decomposition; microbial inactivation is achieved through the synergistic effect of ultra-high temperature instantaneous treatment and vacuum flash cooling. In the enzymatic hydrolysis process, the pretreated liquid material is accelerated by a high-pressure plunger pump and atomized into micron-sized droplets through a spiral microchannel nozzle. During the spraying process, heat exchange occurs with superheated steam. The steam volume is dynamically adjusted by detecting changes in the material's transmittance, and blockchain technology is used to record the sterilization temperature-time curve. Finally, the liquid is converted into a solid state through a spray drying tower.

[0049] This embodiment has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention.

[0050] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An enzymatic hydrolysis process for preparing compound enzymes, characterized in that, Includes the following steps: S1. Strain selection and genetic engineering optimization to modify enzyme-producing strains and enhance the expression of the target enzyme encoding gene; S2. High-density fermentation culture is carried out using fed-batch fermentation technology; S3. Multi-stage separation and purification: The purification process includes centrifugation and ion chromatography. Inside the centrifuge tank, the solution concentrates to the side due to centrifugation, generating pressure. The enzyme is then transported through a filter membrane to the bottom ion chromatography unit for chromatographic purification. Selective adsorption and separation are achieved through electrostatic interaction between the oppositely charged chromatographic channels and the target enzyme. The enzyme preparation raw materials are injected into the centrifuge unit through the top injection pipe. The motor in the drive mechanism is started, driving the drive shaft to rotate. The top of the drive shaft, via a second gear, drives the toothed belt to rotate, which in turn drives the central shaft at the bottom to rotate. When the central shaft rotates, the guide plates at the top and bottom pull the scraper embedded inside to rotate. When the scraper rotates at a slower speed, it pushes the flowing enzyme solution, helping the enzyme solution in the center to contact the chromatography medium on the inner wall of the chromatography channel. After completing the chromatography purification, by increasing the speed of the motor, the speed of the central shaft is increased, causing the scraper to generate greater centrifugal force. At this time, the magnetic column on the inner side of the scraper can be separated from the magnetic sleeves on both sides, so that the scraper can directly press against the inner wall of the chromatography channel. Then, in the subsequent rotation process, the enzyme solution adhering to the chromatography medium is directly scraped off, completing the enzyme purification process. S4. Material mixing: After the pre-treated crushed raw material is fed into the mixing chamber through a screw conveyor belt, the material is subjected to a triple action of shearing, impact and cavitation effect under high pressure. S5. Continuous enzymatic hydrolysis is carried out using a jacketed stirred reaction vessel. The phase change material in the jacket layer absorbs the heat of reaction, and the temperature is controlled by a semiconductor temperature control module. S6. Flash sterilization treatment deactivates enzymes and prevents excessive decomposition.

2. The enzymatic hydrolysis process for preparing compound enzymes according to claim 1, characterized in that: In step S1, by integrating gene editing and directed evolution technologies, the enzyme-producing strain is precisely modified to target and enhance the expression of the gene encoding the target enzyme, and a high-throughput screening platform is used to screen out highly efficient mutant strains that are resistant to extreme environments.

3. The enzymatic hydrolysis process for preparing compound enzymes according to claim 2, characterized in that, In step S2, a real-time online sensor control system integrating dissolved oxygen, carbon-nitrogen ratio and inducer concentration is integrated, and metal ions such as Ca²⁺ and Mg²⁺ are added as cofactors to activate the enzyme protein folding active center. The accumulation of metabolic byproducts was inhibited by a glucose gradient-limited feeding strategy.

4. The enzymatic hydrolysis process for preparing compound enzymes according to claim 1, characterized in that: The toothed belt drives the injection pipe in the centrifuge assembly to rotate, thereby causing the bottom linkage shaft and push plate to rotate at high speed inside the centrifuge tank.

5. The enzymatic hydrolysis process for preparing compound enzymes according to claim 4, characterized in that: The enzyme solution enters the ion chromatography assembly and adsorbs onto the chromatography medium on the inner wall of the chromatography channel. During this process, the motor in the drive mechanism is activated, which drives the drive shaft to rotate. The drive shaft drives the surface gear to rotate, and the drive gear is embedded in the fixed track, driving the rotating ring inside the fixed track. With the help of the outer and inner gear rings, each fourth gear is controlled to rotate, so that each ion chromatography assembly can rotate.

6. The enzymatic hydrolysis process for preparing compound enzymes according to claim 1, characterized in that: In S6, the liquid material after microbial inactivation pretreatment is accelerated by a high-pressure plunger pump through the synergistic effect of ultra-high temperature instantaneous treatment and vacuum flash cooling, and atomized into micron-sized droplets through a spiral microchannel nozzle, and heat exchange with superheated steam during the spraying process.

7. The enzymatic hydrolysis process for preparing compound enzymes according to claim 6, characterized in that: The amount of steam is dynamically adjusted by detecting changes in the light transmittance of the material, and the sterilization temperature-time curve is recorded using blockchain technology. Finally, the liquid is converted into a solid state through a spray drying tower.

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