Passivating enzyme soluble concentrate and preparation process thereof

Abstract

The invention relates to the technical field of biological agents, and discloses a passivating enzyme soluble concentrate and a preparation process thereof.The soluble concentrate takes a specially-designed multifunctional deep eutectic solvent as a unique carrier, and the solvent has the multiple functions of stabilizing enzyme conformation, resisting oxidation, providing plant metabolism and increasing energy and the like. An active component is composite passivating enzyme powder which is subjected to vacuum freeze drying, and is compounded with a plant-derived phloem conduction enhancer in a synergetic manner. The preparation process adopts a core strategy of curing first and then dissolving, and the enzyme freeze-dried powder is dispersed in the non-aqueous solvent under low-temperature mild conditions. By constructing a water-free environment and adopting an activity protection process, the problems that a traditional water-based enzyme preparation is unstable in storage and the activity is easy to lose are fundamentally solved, and meanwhile, the bioavailability is remarkably improved by virtue of a targeted conduction component. The preparation has the remarkable advantages of ultra-long stability at normal temperature, high initial enzyme activity and strong targeting property.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to an enzyme-inactivating soluble agent and its preparation process. Background Technology

[0002] Currently, vascular diseases of plants caused by soil-borne pathogens such as *Fusarium oxysporum* and *Verticillium wiltii* are a persistent problem in global agricultural production. After infecting plant roots, these pathogens multiply in the xylem or phloem, physically blocking xylem vessels and sieve tubes due to their mycelium and secreted metabolites, such as polysaccharides. This severely hinders the normal transport of water and nutrients within the plant, ultimately leading to wilting, yellowing, and even death, causing significant economic losses. Utilizing bio-enzymes to degrade these blockages and clear the vascular bundles is considered a promising technological approach to solving these diseases.

[0003] In response to the aforementioned issues, several bio-enzyme preparations have been developed in the prior art. Their preparation typically involves preliminary purification of the enzyme stock solution obtained from fermentation, followed by dilution and compounding in an aqueous buffer system. To maintain short-term stability, pH adjusters, conventional humectants (such as glycerol), or ion chelating agents are added to the aqueous solution. The final product is usually in the form of an aqueous solution or suspension. When used, this water-based enzyme preparation is diluted and applied to crops via root irrigation or injection, with the expectation that the enzyme molecules can enter the plant, break down the blockages caused by pathogens, and restore transport function.

[0004] However, in long-term practice, existing technologies have revealed their inherent limitations. First, enzyme stability is a core weakness. Enzyme proteins are essentially biological macromolecules, and their activity is highly dependent on their specific spatial structure. In traditional water-based formulations, the ubiquitous water molecules continuously induce slow hydrolysis of the enzyme protein, fundamentally determining that its activity will decay over time. This instability results in a very short shelf life and often requires stringent cold chain conditions for storage and transportation, significantly increasing costs.

[0005] Secondly, conventional preparation processes themselves may damage enzyme activity. In order to dissolve or disperse various auxiliaries evenly, the preparation process may involve high-speed shearing or gentle heating. These operations are severe challenges to the fragile three-dimensional structure of enzymes, often resulting in a significant reduction in the initial activity of the finished product before it leaves the factory.

[0006] Finally, even when enzymes are successfully formulated, their effectiveness in plants is often unsatisfactory. How to efficiently penetrate the layers of physiological barriers and be precisely delivered to the vascular tissues where blockage has actually occurred after the active ingredients are applied to the roots remains a challenge that current technologies have not effectively addressed. The lack of an active targeting mechanism means that most of the active ingredients are lost or degraded before reaching their sites of action, resulting in field efficacy far below expectations. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a soluble inactivating enzyme and its preparation process, aiming to solve the technical problems of existing inactivating enzyme preparations, which suffer from short shelf life, high application costs, and poor field efficacy due to unstable storage, damage during preparation, and low translocation efficiency in plants.

[0008] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of the present invention provides an enzyme-inactivating soluble concentrate, which, by mass parts, comprises: 1-8 parts of a compound enzyme-inactivating lyophilized powder; 0.2-2 parts of a plant-derived phloem conduction enhancer; 0.5-4 parts of a bio-derived penetration enhancer; and 86-98.3 parts of a multifunctional deep eutectic solvent.

[0009] In this invention, a specially designed multifunctional deep eutectic solvent is used as a non-aqueous carrier for the enzyme and compounded with enzyme powder that has undergone vacuum freeze-drying to construct a stable system that is essentially free of free water. This multifunctional deep eutectic solvent, through its dense hydrogen bond network structure, confines and inactivates the spatial conformation of the enzyme at the molecular level. Simultaneously, its extremely low water activity effectively inhibits enzyme hydrolysis and inactivation, as well as microbial growth, thus solving the stability problem caused by the high water activity in traditional aqueous formulations.

[0010] The multifunctional deep eutectic solvent is composed of four components: a hydrogen bond acceptor, a first hydrogen bond donor, a second hydrogen bond donor, and a redox buffer pair. The hydrogen bond acceptor is choline chloride, the first hydrogen bond donor is L-lactic acid, and the second hydrogen bond donor is L-malic acid. All three components are plant-derived or plant-metabolic compatible substances. L-lactic acid and L-malic acid can serve as intermediate metabolites in the plant tricarboxylic acid cycle (TCA), while choline chloride serves as a substance required for plant growth. This allows the solvent itself to not only act as a stabilizing carrier but also to be compatible with metabolic processes within the plant.

[0011] The redox buffer pair in the multifunctional eutectic solvent is selected from at least one of a mixture of L-glutathione (reduced) and L-glutathione (oxidized), or a mixture of L-cysteine ​​and L-cysteine. The introduction of this redox buffer pair endows the solvent system with redox potential buffering capacity, protecting the tertiary structure of the complex inactivating enzyme from oxidative stress and further enhancing the chemical stability of the formulation during storage and use.

[0012] In the multifunctional deep eutectic solvent, the molar ratio of choline chloride, L-lactic acid, L-malic acid, and the redox buffer pair is 1:(1.2-1.8):(0.4-0.8):(0.05-0.2). This specific molar ratio range ensures that the components can form a homogeneous, stable deep eutectic liquid with suitable viscosity, and can effectively dissolve and stabilize the lyophilized compound inactivating enzyme powder.

[0013] This invention also introduces plant-derived phloem conduction enhancers, such as sucrose fatty acid esters or methyl salicylate, to promote the lateral transport and long-distance conduction of active components from the xylem to the phloem within the plant, thereby improving the efficiency of the active components reaching the target site (sieve tubes). Simultaneously, by introducing bio-derived penetration enhancers, such as alkyl glycosides, the surface tension of the solution is reduced, improving its spreading and penetration properties at the plant tissue interface.

[0014] A second aspect of the present invention provides a preparation process for the above-mentioned inactivating enzyme soluble agent, the preparation process comprising the following steps: S1. Preparation of multifunctional deep eutectic solvent: Hydrogen bond acceptor, first hydrogen bond donor, second hydrogen bond donor and redox buffer are mixed, heated and stirred until a homogeneous and clear liquid is formed, and then cooled to obtain multifunctional deep eutectic solvent. S2. Enzyme solidification: The aqueous composite inactivation enzyme stock solution is freeze-dried under vacuum to obtain composite inactivation enzyme lyophilized powder. S3. Low-temperature dissolution: Under low-temperature conditions, the composite inactive enzyme lyophilized powder obtained in step S2 is dissolved in the multifunctional deep eutectic solvent prepared in step S1. S4. Compounding and homogenization: Add plant-derived phloem conduction enhancer and bio-derived permeability enhancer to the mixture in step S3, stir evenly, and obtain the inactivating enzyme soluble solution.

[0015] The core of this preparation process lies in the adoption of a "solidification-then-dissolution" technical route. Step S2 separates the enzyme from the unstable aqueous environment to form a stable solid powder. Step S3 then redissolves the solid enzyme powder in a non-aqueous eutectic solvent under low-temperature conditions. This process avoids phase separation or enzyme protein denaturation that may occur when directly mixing aqueous enzyme solutions with eutectic solvents, thus maximizing the protection of the enzyme's biological activity.

[0016] The heating temperature in step S1 is set to 65℃-75℃ and carried out under inert gas protection. This temperature range effectively promotes the formation of a deep eutectic solvent, while the inert gas protection prevents the oxidation of components during the heating process.

[0017] In step S2, the shelf temperature range for vacuum freeze drying is -50°C to 25°C, and the vacuum degree is less than 15Pa, to ensure sufficient removal of moisture and effective preservation of enzyme activity.

[0018] The low-temperature conditions in step S3 are 10℃-20℃. Dissolving at this temperature slows down molecular thermal motion, which helps maintain the native conformation of the enzyme protein in a high-viscosity system.

[0019] In step S1, the hydrogen bond acceptor is choline chloride, the first hydrogen bond donor is L-lactic acid, and the second hydrogen bond donor is L-malic acid.

[0020] This invention provides an enzyme-inactivating soluble agent and its preparation process. It has the following beneficial effects: 1. This invention employs a specially designed multifunctional deep eutectic solvent, replacing the traditional aqueous carrier. The preparation process incorporates enzyme powder treated with vacuum freeze-drying technology, creating a formulation environment virtually free of free water. This design fundamentally inhibits the enzyme's hydrolytic inactivation pathway, and also hinders microbial growth. Existing water-based formulations generally suffer from easy loss of activity and reliance on cold chain transportation. This invention solves this problem, achieving long-term stable storage of the product at room temperature and significantly reducing application costs.

[0021] 2. This invention employs a unique "solid-first, then dissolve" preparation process. First, the aqueous enzyme solution is prepared into a highly active lyophilized powder. Then, under stringent low-temperature and low-shear conditions, the enzyme powder is dissolved in a non-aqueous solvent. Unlike existing technologies that directly mix the enzyme stock solution or use heating to promote dissolution, this process cleverly avoids the damage to the enzyme protein structure caused by thermal stress and phase transition shocks. This method solves the technical pain point of easy activity loss during preparation, ensuring that the final product has extremely high initial biological activity, laying the foundation for its efficacy.

[0022] 3. This invention endows the solvent system with synergistic effects. The main components of the solvent, such as L-lactic acid and L-malic acid, are bioactive substances that can be absorbed by plants. The introduced redox buffer pair endows the system with the ability to resist oxidative stress. This changes the limitation of the carrier in existing technologies, which merely serves as an inert dispersion medium. Through this "active carrier" design, this invention not only enhances the chemical stability of enzymes during storage and application but also provides additional physiological support for plants, solving the problem of limited effects of single functional components and achieving synergistic effects between components.

[0023] 4. This invention constructs a highly efficient in vivo targeted delivery system by incorporating a plant-derived phloem conduction enhancer into the composition. This component can actively guide the transport of inactivating enzyme active ingredients within the plant. It promotes the movement and accumulation of active substances from the application site to the target site—the vascular bundle sieve tubes. Existing technologies often suffer from poor efficacy due to the inefficient delivery of active ingredients to lesions. This invention solves the problem of low transport efficiency of active ingredients within the plant, significantly improving bioavailability and precision of action, resulting in superior control effects in practical applications. Attached Figure Description

[0024] Figure 1 This is a flowchart of the preparation method of the present invention. Detailed Implementation

[0025] The technical solutions in 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.

[0026] Please see the appendix Figure 1 : Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0027] Example 1 1. Composition Formulation The components, by mass parts, are as follows: Lyophilized compound inactivated enzyme powder: 4.0 parts Plant-derived phloem conduction enhancer (sucrose fatty acid ester): 1.0 part Bio-derived penetration enhancer (alkyl glycoside): 2.0 parts Multifunctional deep eutectic solvent (MF-DES): 93.0 parts 2. Preparation steps (1) Preparation of Multifunctional Deep Eutectic Solvent (MF-DES): A mixture of choline chloride, L-lactic acid, L-malic acid, and L-glutathione (reduced / oxidized form) was accurately weighed according to a molar ratio of 1:1.5:0.6:0.1. All weighed components were added to a dry reaction vessel, and nitrogen gas was introduced for protection. Stirring was started, and the temperature of the reaction vessel was raised to 70°C and maintained. Stirring was continued until all solids were completely dissolved, forming a homogeneous, clear, viscous liquid. Heating was stopped, and the mixture was cooled to room temperature with stirring to obtain the multifunctional deep eutectic solvent.

[0028] (2) Preparation of the composite inactivating enzyme lyophilized powder: The aqueous solution of the composite inactivating enzyme was pre-frozen at -45℃. The vacuum system was started, and the pressure in the drying chamber was reduced to 10Pa. Then, the shelf temperature was raised to -20℃ for a first sublimation drying, followed by a second desorption drying at 20℃. After drying, the vacuum was broken to obtain the composite inactivating enzyme lyophilized powder with a water content of less than 2.0%, which was then sealed for later use.

[0029] (3) Preparation of the finished product: Transfer 93.0 parts of MF-DES obtained in step (1) to a low-temperature preparation vessel and control the temperature inside the vessel at 15℃. Turn on gentle stirring and slowly add 4.0 parts of the compound inactivating enzyme lyophilized powder obtained in step (2) into the vessel, stirring until completely dissolved. Then, add 1.0 parts of sucrose fatty acid ester and 2.0 parts of alkyl glycoside in sequence, and continue stirring for 30 minutes until the system is uniformly mixed. Finally, adjust the pH value to 6.5 with potassium hydroxide solution to obtain a homogeneous and clear inactivating enzyme soluble solution.

[0030] Example 2 1. Composition Formulation The components, by mass parts, are as follows: Compound inactivated enzyme lyophilized powder: 1.0 part Plant-derived phloem conduction enhancer (methyl salicylate): 0.2 parts Bio-derived penetration enhancer (alkyl glycoside): 0.5 parts Multifunctional deep eutectic solvent (MF-DES): 98.3 parts 2. Preparation steps (1) Preparation of Multifunctional Deep Eutectic Solvent (MF-DES): Choline chloride, L-lactic acid, L-malic acid, and a mixture of L-cysteine / L-cysteine ​​were accurately weighed according to a molar ratio of 1:1.2:0.4:0.05. All weighed components were added to a dry reaction vessel, and nitrogen gas was introduced for protection. Stirring was started, and the temperature of the reaction vessel was raised to 65°C and maintained. Stirring was continued until all solids were completely dissolved, forming a homogeneous, clear, viscous liquid. Heating was stopped, and the mixture was cooled to room temperature with stirring to obtain the multifunctional deep eutectic solvent.

[0031] (2) Preparation of the composite inactivating enzyme lyophilized powder: The aqueous solution of the composite inactivating enzyme was pre-frozen to -50℃. The vacuum system was started, and the pressure in the drying chamber was reduced to 12Pa. Then, the shelf temperature was raised to -25℃ for a first sublimation drying, followed by a second desorption drying at 15℃. After drying, the vacuum was broken to obtain the composite inactivating enzyme lyophilized powder with a water content of less than 2.0%, which was then sealed for later use.

[0032] (3) Preparation of the finished product: Transfer 98.3 parts of MF-DES obtained in step (1) to a low-temperature preparation vessel and control the temperature inside the vessel at 10℃. Turn on gentle stirring and slowly add 1.0 part of the composite inactivating enzyme lyophilized powder obtained in step (2) into it, stirring until completely dissolved. Then, add 0.2 parts of methyl salicylate and 0.5 parts of alkyl glycoside in sequence, and continue stirring for 40 minutes until the system is mixed evenly. Finally, adjust the pH value to 6.0 with potassium hydroxide solution to obtain a homogeneous and clear inactivating enzyme soluble solution.

[0033] Example 3 1. Composition Formulation The components, by mass parts, are as follows: Compound inactivated enzyme lyophilized powder: 8.0 parts Plant-derived phloem conduction enhancer (sucrose fatty acid ester): 2.0 parts Bio-derived penetration enhancer (alkyl glycoside): 4.0 parts Multifunctional deep eutectic solvent (MF-DES): 86.0 parts 2. Preparation steps (1) Preparation of Multifunctional Deep Eutectic Solvent (MF-DES): A mixture of choline chloride, L-lactic acid, L-malic acid, and L-glutathione (reduced / oxidized form) was accurately weighed according to a molar ratio of 1:1.8:0.8:0.2. All weighed components were added to a dry reaction vessel, and nitrogen gas was introduced for protection. Stirring was started, and the temperature of the reaction vessel was raised to 75°C and maintained. Stirring was continued until all solids were completely dissolved, forming a homogeneous, clear, viscous liquid. Heating was stopped, and the mixture was cooled to room temperature with stirring to obtain the multifunctional deep eutectic solvent.

[0034] (2) Preparation of the composite inactivating enzyme lyophilized powder: The aqueous solution of the composite inactivating enzyme was pre-frozen to -40℃. The vacuum system was started, and the pressure in the drying chamber was reduced to 8Pa. Then, the shelf temperature was raised to -15℃ for a first sublimation drying, and then the temperature was raised to 25℃ for a second desorption drying. After drying, the vacuum was broken to obtain the composite inactivating enzyme lyophilized powder with a water content of less than 2.0%, which was sealed for later use.

[0035] (3) Preparation of the finished product: Transfer 86.0 parts of MF-DES obtained in step (1) to a low-temperature preparation vessel and control the temperature inside the vessel at 20℃. Turn on gentle stirring and slowly add 8.0 parts of the composite inactivating enzyme lyophilized powder obtained in step (2) into it, stirring until completely dissolved. Then, add 2.0 parts of sucrose fatty acid ester and 4.0 parts of alkyl glycoside in sequence, and continue stirring for 20 minutes until the system is mixed evenly. Finally, adjust the pH value to 7.0 with potassium hydroxide solution to obtain a homogeneous and clear inactivating enzyme soluble solution. Comparative Example 1: Compared with Example 1, the difference is that 93.0 parts of the multifunctional deep eutectic solvent were replaced with 93.0 parts of deionized water, and 4.0 parts of the compound inactivating enzyme lyophilized powder were replaced with an equal amount of compound inactivating enzyme aqueous solution stock solution with enzyme activity. All other aspects are the same.

[0036] Comparative Example 2: Compared with Example 1, the difference is that when preparing the multifunctional deep eutectic solvent, a mixture of L-glutathione (reduced / oxidized) with a molar ratio of 0.1 is not added, that is, the prepared multifunctional deep eutectic solvent does not contain a redox buffer pair, and all other aspects are the same.

[0037] Comparative Example 3: Compared with Example 1, the difference is that: 1.0 part of plant-derived phloem conduction enhancer (sucrose fatty acid ester) is not added to the finished product formulation. Its mass part is made up by the multifunctional deep eutectic solvent, that is, the amount of multifunctional deep eutectic solvent is adjusted to 94.0 parts, and the rest are the same.

[0038] Comparative Example 4: Compared with Example 1, the difference is that in the finished product preparation step (3), the temperature at which the composite inactive enzyme lyophilized powder is dissolved in the multifunctional deep eutectic solvent is changed from 15°C to 70°C, while the rest are the same.

[0039] Comparative Example 5: Compared with Example 1, the difference is that when preparing the multifunctional deep eutectic solvent, the molar ratio of its components is changed to 1:2.5:0.6:0.1, that is, the molar number of the first hydrogen bond donor L-lactic acid exceeds its limited range (1.2-1.8), while the rest are the same.

[0040] Comparative Example 6: Compared with Example 1, the difference is that step (2) is not used to freeze-dry the composite inactivating enzyme stock solution under vacuum. Instead, an equal amount of composite inactivating enzyme aqueous solution stock solution with enzyme activity is directly added to a multifunctional deep eutectic solvent for mixing. All other aspects are the same.

[0041] Experiment 1: Comparative Test of Formulation Storage Stability 1. Experimental Objective This test aims to compare the activity retention of the inactivation enzyme preparations of Example 1 (based on a multifunctional deep eutectic solvent) and Comparative Example 1 (based on traditional deionized water) at different time points through room temperature storage and accelerated high temperature storage experiments, thereby quantitatively evaluating the beneficial effect of the non-aqueous system of the present invention in improving product storage stability.

[0042] 2. Experimental Samples Experimental group: Inactivating enzyme soluble solution prepared according to the method in Example 1.

[0043] Control group: Inactivating enzyme aqueous solution prepared according to the method of Comparative Example 1.

[0044] 3. Experimental Procedure (1) Sample dispensing and grouping: The prepared experimental and control group samples were dispensed into several identical brown screw-top bottles of good airtightness, 50 mL per bottle, and sealed. The two groups of samples were then divided into two batches for room temperature storage and accelerated high temperature storage tests, respectively.

[0045] (2) Storage conditions settings: Batch storage at room temperature: Place a batch of samples from the experimental group and the control group in a constant temperature incubator at 25℃±2℃ and store them in the dark.

[0046] Accelerated high-temperature storage batches: Another batch of samples from the experimental group and the control group were placed in a constant-temperature incubator at 40℃±2℃ and stored in the dark.

[0047] (3) Periodic sampling and activity determination: On days 0, 30, 90, and 180 of the start of storage, three parallel samples were taken from each of the experimental and control groups under each storage condition (25℃ and 40℃). The activity of the complex inactivating enzyme in the samples was determined by spectrophotometry. The rate of change of absorbance at the measurement wavelength was recorded, and the actual enzyme activity units (U / mL) of each sample were calculated according to the standard curve.

[0048] (4) Data processing and calculation: For each sample group at each time point, the average value of the enzyme activity measurements from three parallel samples was taken. The enzyme activity retention rate was calculated according to the following formula: ; 4. Experimental Data Recording The experimental data obtained from the above experiment are recorded in the table below.

[0049] Table 1: Comparison of the retention rate of compound inactivating enzyme activity in samples under different storage conditions (%) Summarize: The test results clearly demonstrate that the enzyme-inactivating soluble concentrate provided by this invention exhibits significant technical advantages in terms of storage stability. According to the data in Table 1, after 180 days of storage at room temperature (25°C), the enzyme activity retention rate of Example 1 remained as high as 95.8%, while under the same conditions, the activity of Comparative Example 1, based on a traditional water-based system, decreased to 73.4%. This difference was even more pronounced under the harsh conditions of accelerated high temperature (40°C), where Example 1 maintained 86.7% activity after 180 days of storage, while the activity of Comparative Example 1 had severely declined, remaining at only 18.9%. This result directly confirms the effectiveness of the technical solution of this invention.

[0050] The aforementioned superior stability stems from the fundamental innovation of this invention regarding the formulation carrier. By employing a specially designed multifunctional eutectic solvent instead of traditional water, a non-aqueous environment essentially free of free water is constructed. In this system, the extremely low water activity fundamentally inhibits the hydrolytic reaction pathways that lead to the degradation of the enzyme protein structure, while also eliminating the moisture conditions required for microbial growth. This is fundamentally different from the situation in Comparative Example 1, where water is used as the solvent, and the enzyme molecules are constantly exposed to a large number of water molecules, making them prone to hydrolysis and inactivation.

[0051] Furthermore, the multifunctional eutectic solvent in this invention is not simply an inert carrier; its own structure actively protects the enzyme molecules. Through the dense, three-dimensional hydrogen bond network formed between its components, the solvent physically confines and supports the three-dimensional conformation of the complex inactivating enzyme at the molecular scale, effectively preventing protein molecule unfolding and denaturation caused by intensified thermal motion (especially at 40°C). Simultaneously, the integrated redox buffer pairs in this solvent system provide continuous chemical protection for the enzyme, effectively neutralizing any oxidizing substances that may be generated in the system. This, combined with the physical confinement effect, constructs a multidimensional stabilization system, ensuring the long-term maintenance of enzyme activity.

[0052] Experiment 2: Effects of preparation process and key components on initial enzyme activity 1. Experimental Objective This test aims to verify, by comparing the initial enzyme activity of the finished product under different preparation conditions, the key roles of the "first solidify, then dissolve" process, the low-temperature dissolution step, and the addition of a redox buffer pair in the solvent system adopted in this invention in maximizing the protection of the bioactivity of the composite inactivating enzyme and obtaining a highly active finished product.

[0053] 2. Experimental Samples Experimental group: Inactivating enzyme soluble solution prepared according to the method in Example 1.

[0054] Control group 1: The sample prepared according to the method of comparative example 2 (which does not contain redox buffer pairs in its multifunctional deep eutectic solvent).

[0055] Control group 2: Samples prepared according to the method of comparative example 4 (dissolved at a high temperature of 70°C).

[0056] Control group 3: Samples prepared according to the method of comparative example 6 (the stock solution of the composite inactivating enzyme was directly mixed with a multifunctional deep eutectic solvent).

[0057] 3. Experimental Procedure (1) Sample preparation and equilibration: Samples for the experimental group and each control group were prepared according to the methods described in Example 1, Comparative Example 2, Comparative Example 4 and Comparative Example 6, respectively. After all samples were prepared, they were allowed to stand at room temperature (25°C) for equilibration for 2 hours.

[0058] (2) Sampling and dilution: Accurately weigh equal masses (e.g., 1.0 g) of sample from each group, repeating 3 times for each group. Place the sample into a volumetric flask pre-filled with 99.0 mL of phosphate buffered saline (PBS, pH 7.0), bring the volume to 100 mL, and shake thoroughly to obtain a 100-fold dilution for subsequent enzyme activity assays. This dilution step aims to reduce the viscosity of the multifunctional eutectic solvent, ensuring that the substrate can fully contact the enzyme molecules.

[0059] (3) Initial enzyme activity determination: The initial enzyme activity of each diluted sample was determined by spectrophotometry. The substrate solution and the diluted sample solution were added to the cuvette in sequence, mixed quickly, and then placed in the spectrophotometer. The change in absorbance was continuously monitored at a specific wavelength. The rate of change in absorbance during the linear phase of the initial reaction was recorded.

[0060] (4) Data processing and calculation: Based on the molar extinction coefficient of the substrate and the following formula, the initial enzyme activity of each sample was calculated and finally converted into enzyme activity units (U / g) per unit mass of sample. The average value of three parallel measurements for each sample group was taken as the final result.

[0061] active Dilution number; in, The rate of change of absorbance. The total volume of the reaction is [volume]. The molar extinction coefficient is . For optical path, This refers to the volume of sample diluent added. This refers to the mass of the original sample.

[0062] 4. Experimental Data Recording The experimental data obtained from the above experiment are recorded in the table below.

[0063] Table 2: Effects of different preparation processes and components on the initial activity of the composite inactivating enzyme (U / g) Summarize: The experimental data in Table 2 clearly demonstrate that the preparation process and key components of this invention play a decisive role in obtaining highly active end products. Example 1, employing the complete technical solution, achieved an initial enzyme activity as high as 485.7 U / g. In contrast, any deviation from key steps or components led to a significant decrease in activity: Comparative Example 6, which directly mixed the aqueous enzyme solution, had an activity of only 112.4 U / g; Comparative Example 4, which used high-temperature dissolution, saw its activity drastically reduced to 165.9 U / g; and Comparative Example 2, lacking a redox buffer pair, also exhibited significantly lower activity than the experimental group. This series of comparisons powerfully demonstrates the necessity and advancement of the specific process and formulation design of this invention.

[0064] The "solidification followed by dissolution" process of this invention is the primary prerequisite for protecting enzyme activity. Comparative Example 6 attempted to directly mix the aqueous enzyme stock solution into a high-concentration eutectic solvent. This operation leads to drastic changes in local water activity and instantaneous shocks to the solvent environment, easily causing enzyme protein aggregation and precipitation, resulting in irreversible inactivation and ultimately extremely low initial activity. In contrast, this invention first converts the enzyme into a stable solid powder through vacuum freeze-drying, and then dissolves it in a non-aqueous solvent under gentle low-temperature conditions. This process avoids the drastic phase transition of direct liquid-liquid mixing, allowing enzyme molecules to be gradually and gently dispersed in the non-aqueous matrix in a controlled environment, thereby maximizing the preservation of its native conformation and biological activity.

[0065] Furthermore, temperature control and the construction of the chemical environment during the preparation process are equally crucial. The three-dimensional structure of enzyme proteins relies on fragile non-covalent bonds for maintenance and is extremely sensitive to thermal stress. As shown in Comparative Example 4, dissolution at a high temperature of 70°C is too hot, enough to destroy these critical structures, leading to rapid denaturation and inactivation of the enzyme. The low-temperature dissolution step (10°C-20°C) used in this invention effectively avoids this thermal damage. Meanwhile, the results of Comparative Example 2 demonstrate the important value of redox buffers. During the preparation process, even with inert gas protection, trace amounts of oxidizing substances can still damage the enzyme's active sites or key amino acid residues. This invention, by pre-constructing a redox buffer system in the solvent, can actively eliminate these potential oxidative threats, providing an additional layer of chemical protection for the enzyme and ensuring that the final product has the highest possible initial biological activity.

[0066] Experiment 3: Comparative Testing of Functional Properties of Synergistic Components and Formulation Systems 1. Experimental Objective This test consists of two parts: first, by observing physical properties, it verifies the necessity of the specific molar ratio of the multifunctional deep eutectic solvent components described in this invention for maintaining the physical stability of the formulation system; second, by conducting potted plant experiments, it verifies the synergistic effect of the plant-derived phloem conduction enhancer added to the formulation of this invention in improving the actual application effect of the formulation.

[0067] 2. Experimental Samples Experimental group: Inactivating enzyme soluble solution prepared according to the method in Example 1.

[0068] Control group 1: Samples prepared according to the method of comparative example 3 (without plant-derived phloem conduction enhancer).

[0069] Control group 2: Samples prepared according to the method of comparative example 5 (the molar ratio of the components of the multifunctional deep eutectic solvent exceeds the limit).

[0070] 3. Experimental Procedure (A) Observation of the physical stability of the formulation Sample preparation: Take 100 mL of freshly prepared samples from Example 1 and Comparative Example 5 respectively, and place them in two clean, dry 100 mL glass colorimetric tubes, then seal the tube openings.

[0071] Observe under static conditions: Place the two colorimetric tubes vertically on a constant temperature experimental platform at 25℃ and let them stand in the dark.

[0072] Record the characteristics: Visually observe and record the appearance of the two samples at 0 hours, 24 hours and 7 days after standing, paying particular attention to whether there is any layering, turbidity, precipitation or crystallization.

[0073] (B) Plant application effect test Test plants and pathogen: Cucumber seedlings of uniform growth, at the 5-6 leaf stage, were selected. Fusarium oxysporum suspension was inoculated into the soil around the roots of each seedling using a root drenching method to induce cucumber wilt (a typical vascular bundle blockage disease). After inoculation, the seedlings were managed routinely in the greenhouse. Treatment with fungicide was initiated when initial wilting symptoms became widespread.

[0074] Grouping and treatment: Cucumber seedlings with similar disease severity were randomly divided into three groups of 20 seedlings each.

[0075] Experimental group: using the formulation of Example 1.

[0076] Control group 1: The formulation used in comparison example 3 was used.

[0077] Blank control group: Apply an equal amount of water. Treatment was carried out by root irrigation, with each preparation diluted 500 times and 100 mL of the diluted solution applied to each seedling.

[0078] Disease assessment and grading: Disease status was assessed on all cucumber seedlings on days 7 and 14 after pesticide application. The disease grading criteria are as follows: Grade 0: The plant shows no symptoms and is growing normally.

[0079] Level 1: The lower 1-2 leaves of the plant wilt at midday, but recover in the morning or evening.

[0080] Grade 2: Most of the plant's leaves are wilted, and the vascular bundles at the base of the stem turn brown.

[0081] Level 3: The entire plant is severely wilted, and most of the leaves are withered and yellow.

[0082] Level 4: Plant dead.

[0083] Data calculation: Based on the disease severity grading results of each group, calculate the disease severity index and prevention and control effect.

[0084] ; Prevention and control effects; 4. Experimental Data Recording The experimental data obtained from the above experiment are recorded in the table below.

[0085] Table 3: Observation Records of Physical Property Stability of Different Formulation Systems Table 4: Control effects of different formulations on cucumber wilt disease.

[0086] Summarize: The test results in Tables 3 and 4, from the perspectives of physical stability and application effect, jointly verify the precision and synergy of the formulation system design of this invention. Physical property observations show that the sample of Example 1, prepared with components in a specific molar ratio, remained homogeneous and clear after long-term standing, while Comparative Example 5, with a molar ratio exceeding this range, exhibited crystallization. This indicates that the solvent system designed in this invention is not a simple mixture of components, but a precisely structured synergistic system.

[0087] The mechanism behind this excellent physical stability lies in the fact that the selected hydrogen bond acceptor and double hydrogen bond donor can only form a deep eutectic system with the most stable and lowest melting point within a specific molar ratio range. Under this "optimal ratio," the components achieve the most efficient intermolecular stacking and interaction through hydrogen bonding, forming a thermodynamically stable homogeneous liquid phase, thereby effectively suppressing the crystallization tendency of any single component. Once this ratio is broken, as shown in Comparative Example 5, excess components cannot fully integrate into the hydrogen bond network and will precipitate from the system under energy-driven conditions, compromising the homogeneity and stability of the formulation.

[0088] Regarding the plant application effects, the data in Table 4 reveals the key value of the synergistic components in this invention. In controlling cucumber wilt, Example 1, containing a plant-derived phloem conduction enhancer, achieved a control effect of 85.9%, while Comparative Example 3, lacking this enhancer, only achieved a control effect of 31.4%. The fundamental reason for this significant difference lies in the fact that this invention not only provides a stable enzyme preparation but also constructs a highly efficient in vivo delivery system. The phloem conduction enhancer actively guides the targeted transport of the enzyme's active ingredients within the plant, enabling them to cross physiological barriers and efficiently enter and distribute from the application site (roots) to their true site of action—the vascular bundle system—effectively clearing blockages caused by pathogen infection. Without this "navigation" component, the active ingredients cannot efficiently reach the lesion, naturally resulting in a significantly reduced application effect.

[0089] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A soluble solution for inactivating enzymes, characterized in that, The components include the following parts by mass: Compound inactivated enzyme lyophilized powder: 1-8 parts; Plant-derived phloem conduction enhancer: 0.2-2 parts; Bio-derived penetration enhancer: 0.5-4 parts; Multifunctional deep eutectic solvent: 86-98.3 parts.

2. The enzyme-inactivating soluble agent according to claim 1, characterized in that, The multifunctional deep eutectic solvent is a non-aqueous solvent prepared by mixing hydrogen bond acceptors, a first hydrogen bond donor, a second hydrogen bond donor, and a redox buffer pair in a preset molar ratio. Wherein, the hydrogen bond acceptor is choline chloride, the first hydrogen bond donor is L-lactic acid, the second hydrogen bond donor is L-malic acid, and the molar ratio of the three is (1.2-1.8):(0.4-0.8):(0.05-0.2).

3. The enzyme-inactivating soluble agent according to claim 2, characterized in that, The redox buffer pair in the multifunctional eutectic solvent is selected from at least one of a mixture of L-glutathione (reduced form) and L-glutathione (oxidized form), or a mixture of L-cysteine ​​and L-cysteine.

4. The enzyme-inactivating soluble agent according to claim 1, characterized in that, The plant-derived phloem conduction enhancer is selected from at least one of sucrose fatty acid esters or methyl salicylate.

5. The enzyme-inactivating soluble agent according to claim 1, characterized in that, The bio-derived penetration enhancer is an alkyl glycoside.

6. A process for preparing an enzyme-inactivating soluble agent, used to prepare the enzyme-inactivating soluble agent according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Preparation of multifunctional deep eutectic solvent: Hydrogen bond acceptor, first hydrogen bond donor, second hydrogen bond donor and redox buffer are mixed, heated and stirred until a homogeneous and clear liquid is formed, and then cooled to obtain multifunctional deep eutectic solvent. S2. Enzyme solidification: The aqueous composite inactivation enzyme stock solution is freeze-dried under vacuum to obtain composite inactivation enzyme lyophilized powder. S3. Low-temperature dissolution: Under low-temperature conditions, the composite inactive enzyme lyophilized powder obtained in step S2 is dissolved in the multifunctional deep eutectic solvent prepared in step S1. S4. Compounding and homogenization: Add plant-derived phloem conduction enhancer and bio-derived permeability enhancer to the mixture in step S3, stir evenly, and obtain the inactivating enzyme soluble solution.

7. The preparation process of an enzyme-inactivating soluble solvent according to claim 6, characterized in that, In step S1, the heating temperature is 65℃-75℃, and the process is carried out under the protection of an inert gas.

8. The preparation process of an enzyme-inactivating soluble solvent according to claim 6, characterized in that, In step S2, the shelf temperature range for vacuum freeze drying is -50°C to 25°C, and the vacuum degree is less than 15Pa.

9. The preparation process of an enzyme-inactivating soluble solvent according to claim 6, characterized in that, In step S3, the low temperature condition is 10℃-20℃.

10. The preparation process of an enzyme-inactivating soluble solvent according to claim 6, characterized in that, In step S1, the hydrogen bond acceptor is choline chloride, the first hydrogen bond donor is L-lactic acid, and the second hydrogen bond donor is L-malic acid.

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