Preparation process of high-activity deoxynivalenol-degrading enzyme

Abstract

The present application relates to the technical fields of enzyme engineering and biological feed additive, and discloses a preparation process of high-activity deoxynivalenol degrading enzyme, which comprises the following steps: firstly, obtaining F428D mutant deoxynivalenol degrading enzyme concentrate in an acetate buffer system; then, under the conditions of stirring and temperature reduction, adding zinc acetate or calcium acetate aqueous solution into the concentrate to perform a coordination reaction; after the reaction is completed, adding trehalose, maltodextrin and pyrroloquinoline quinone disodium salt as protective agents, stirring and dissolving to form a to-be-dried enzyme solution; finally, setting the air inlet temperature to 140-160 DEG C to perform spray drying, and collecting dry powder to obtain the product. The present application solves the problem that the enzyme is easy to be inactivated in spray drying by using the metal ion same ion effect and coordination effect under the acetate buffer environment and combining with specific auxiliary embedding, and has the advantages of short production cycle, low cost, high enzyme activity recovery rate and the like, and is suitable for large-scale industrialized preparation.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering and biological feed additive technology, specifically to a preparation process for a highly active deoxynivalenol-degrading enzyme from Fusarium oxysporum. Background Technology

[0002] Deoxynivalenol (DOX) is widely found in grain feed contaminated with Fusarium, posing a serious threat to livestock health and food safety. Utilizing microbial dehydrogenases or degrading enzymes to convert the toxin into non-toxic products is currently an effective method for removing these mycotoxins in the feed industry. Among these, the DOX degrading enzyme derived from *Youhaiella tibetensis* has attracted attention due to its strong catalytic specificity; however, in practical industrial applications, the formulation processing and activity maintenance of this enzyme preparation face many challenges.

[0003] Enzyme proteins, as biological macromolecules, are highly sensitive to heat and shear stress. While liquid enzyme preparations are simple to prepare in industrial production, they suffer from poor storage stability at room temperature, high transportation costs, and susceptibility to microbial contamination. In contrast, solid enzyme preparations offer advantages such as easy transportation, long shelf life, and ease of compounding with other feed additives. Currently, freeze-drying technology is commonly used in laboratory or small-scale production to prepare solid enzymes. Although this method effectively preserves enzyme activity, it suffers from high energy consumption, long production cycles, and low output, making it difficult to meet the demands of large-scale, low-cost, continuous production of feed additives.

[0004] Spray drying is the preferred method for preparing solid powders in industry, characterized by high throughput and low cost. However, the high-temperature air intake and shear stress during atomization in spray drying can easily disrupt the three-dimensional structure of deoxynivalenol degrading enzymes, leading to enzyme protein denaturation and inactivation. While existing conventional protective agents such as starch and dextrin can provide some physical encapsulation, they cannot provide sufficient rigid support for the enzyme's active site and key structural domains at the molecular level, resulting in generally low enzyme activity recovery rates after spray drying. Therefore, developing a preparation method that can adapt to the spray drying process and effectively maintain the structural stability of the enzyme protein is of great significance for achieving low-cost industrial application of this enzyme. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a preparation process for a highly active deoxynivalenol degrading enzyme from Fusarium nivale, which solves the problem of enzyme inactivation during spray drying.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a preparation process for a highly active deoxynivalenol-degrading enzyme from Fusarium nivale, comprising the following steps: S1. Take the concentrated solution of F428D mutant deoxynivalenol degrading enzyme in an acetate buffer system, and add an aqueous solution of metal acetate dropwise under stirring to carry out a coordination reaction; the metal acetate is selected from any one of zinc acetate and calcium acetate or a mixture thereof. S2. Add trehalose, maltodextrin and disodium pyrroloquinoline quinone to the enzyme solution after the reaction in step S1, stir to dissolve, and obtain the enzyme solution to be dried. S3. Spray dry the enzyme solution to be dried, collect the dry powder, and the target enzyme preparation product is obtained.

[0007] Preferably, the temperature of the coordination reaction in step S1 is controlled between 4 and 10 degrees Celsius; the reaction time is between 2 and 4 hours; and the molar ratio of the metal ions in the acetate metal salt to the enzyme protein in the enzyme concentrate is between 20:1 and 60:1.

[0008] Preferably, the amount of trehalose added in step S2 is 3% to 8% of the enzyme solution mass in step S1, and the amount of maltodextrin added is 10% to 20% of the enzyme solution mass in step S1.

[0009] Preferably, the control parameters for spray drying in step S3 are: inlet air temperature of 140°C to 160°C, outlet air temperature of 65°C to 75°C, and atomization pressure of 0.2 MPa to 0.4 MPa.

[0010] Preferably, the preparation method of the F428D mutant deoxynivalenol degrading enzyme concentrate in the acetate buffer system in step S1 includes: collecting the fermentation supernatant containing the target enzyme, concentrating it by tangential flow ultrafiltration using a membrane with a molecular weight cutoff of 10 kDalton, and then dialyzing the concentrate with a constant volume using an acetate-sodium acetate buffer solution of 50 mmol / L until the difference between the conductivity of the permeate and the conductivity of the buffer solution is less than 5%.

[0011] Preferably, the pH of the acetate-sodium acetate buffer solution is 6.0; and the protein concentration in the F428D mutant deoxynivalenol degrading enzyme concentrate is adjusted to 10 mg / mL.

[0012] Preferably, the amino acid sequence of the F428D mutant deoxynivalenol degrading enzyme is shown in SEQ ID NO.4, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.3.

[0013] Preferably, the F428D mutant deoxynivalenol degrading enzyme concentrate is obtained by fermenting recombinant Pichia pastoris, wherein the recombinant Pichia pastoris comprises an expression vector constructed by linking the gene fragment shown in SEQ ID NO. 3 to the pPICZαA vector and introducing it into Pichia pastoris X-33 competent cells.

[0014] Preferably, the fermentation process includes: adding methanol to induce expression when the wet weight of the cells reaches 200 g / L, and adding a solution of disodium pyrroloquinoline quinone during the induction period to maintain the final concentration of disodium pyrroloquinoline quinone in the fermentation broth at 30 μmol / L.

[0015] Preferably, the aqueous solution of the metal acetate in step S1 is a 1.0 mol / L aqueous solution of zinc acetate dihydrate or a 1.0 mol / L aqueous solution of calcium acetate monohydrate; the molecular weight of the enzyme protein in the enzyme concentrate is 54 kDaltons.

[0016] This invention provides a preparation process for a highly active deoxynivalenol-degrading enzyme from Fusarium nivale. It has the following beneficial effects: 1. This invention utilizes the F428D site-directed mutagenesis to introduce polar aspartic acid residues onto the enzyme protein surface, combined with a specific acetate buffer system, to achieve effective modification of the enzyme molecule by metal ions. In an acetate buffer environment, the added zinc acetate or calcium acetate can remain stable due to the common ion effect and form coordination bonds with the F428D site and adjacent regions. This coordination enhances the structural rigidity of the enzyme protein in solution, providing a structural basis for resisting the thermal shear forces during spray drying, thereby improving the mutant enzyme's tolerance to thermal processing environments.

[0017] 2. This invention significantly improves the drying survival rate of enzyme preparations through the synergistic effect of metal ion coordination pretreatment and composite excipient encapsulation. Trehalose and maltodextrin, as protective agents, can replace water molecules to form hydrogen bonds with polar groups on the enzyme protein surface during high-temperature dehydration, preventing protein denaturation; while pre-treatment with zinc or calcium ion coordination further limits the excessive unfolding of enzyme molecules. The combined use of these two methods allows the enzyme preparation to maintain a high enzyme activity recovery rate even after spray drying at an inlet air temperature of 140-160 degrees Celsius, solving the technical problem of easy inactivation of deoxynivalenol degrading enzymes during hot drying processes.

[0018] 3. This invention directly replaces the fermentation broth with an acetate buffer system using tangential flow ultrafiltration technology, which removes fermentation byproducts and free cofactors, and directly constructs a homogeneous medium environment suitable for metal ion reactions. This process route abandons the high-energy-consuming and long-cycle freeze-drying method, successfully applying spray drying technology to the production of this type of heat-sensitive enzyme preparation, reducing production costs, shortening the preparation cycle, and making it more suitable for large-scale industrial production. Attached Figure Description

[0019] Figure 1 This is a compatibility comparison diagram between the reaction system of this invention and commonly used systems in the prior art; Figure 2This is a box plot showing the metal-binding ability of different enzyme molecular structures in this invention; Figure 3 These are the enzyme activity decay kinetic curves of various embodiments and comparative examples of the present invention under simulated feed pelleting thermal shock. Figure 4 This is a stability trend graph of the enzyme preparation of the present invention under accelerated aging conditions; Figure 5 This is a comparison diagram of the substrate degradation kinetics characteristics of the enzyme preparation of this invention and the comparative example. Detailed Implementation

[0020] 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.

[0021] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0022] Zinc acetate dihydrate (CAS No.: 5970-45-6); Calcium acetate monohydrate (CAS No.: 5743-26-0); Disodium pyrroloquinoline quinone (CAS No.: 122628-50-6) with a purity ≥98.0%; Deoxynivalenol (CAS No.: 51481-10-8) purity ≥98.0%; Trehalose (CAS No.: 6138-23-4); Maltodextrin (CAS No.: 9050-36-6), glucose equivalent DE value controlled at 10-15; Pichia pastoris X-33 and expression vector pPICZαA are both commercially available products. Other conventional chemical reagents, such as acetic acid and sodium acetate, are commercially available analytical grade products.

[0023] Preparation Example 1: This preparation example provides a method for preparing a concentrated solution of F428D mutant deoxynivalenol degrading enzyme in an acetate buffer system, comprising the following steps: 1. Using the wild-type dehydrogenase gene from *Youhaiella tibetensis* as a template, a recombinant wild-type gene fragment with a C-terminal 6xHis tag was designed and synthesized (its nucleotide and amino acid sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively). Site-directed mutagenesis was performed using overlap extension polymerase chain reaction (SEQ ID NO. 3 and SEQ ID NO. 4) to obtain a mutated gene fragment. The beneficial mutant F428D has the recombinant nucleotide and amino acid sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively. This mutated gene fragment was ligated into the *Pichia pastoris* expression vector pPICZαA, electroporated into *Pichia pastoris* X-33 competent cells, and high-copy recombinant strain Re-DON-F428D was obtained by bleomycin plate screening.

[0024] 2. The recombinant strain was inoculated into a basic salt medium for fed-batch fermentation. The fermentation temperature was controlled at 30 degrees Celsius, the pH value was 6.0, and the dissolved oxygen was maintained above 30%. When the wet weight of the cells reached 200 g / L, methanol was fed to induce expression. During the induction period, pyrroloquinoline quinone disodium salt solution was added to the fermentation broth to maintain the final concentration of pyrroloquinoline quinone disodium salt in the fermentation broth at 30 μmol / L. The induction fermentation time was 80 hours.

[0025] 3. After fermentation, the cell bodies were removed by centrifugation, and the fermentation supernatant was collected. Tangential flow ultrafiltration was performed using a polyethersulfone membrane with a molecular weight cutoff of 10 kDa to concentrate the supernatant to one-tenth of its original volume. Subsequently, the concentrate was subjected to constant volume dialysis and filtration using 50 mmol / L acetate-sodium acetate buffer (pH 6.0) until the difference between the conductivity of the permeate and the conductivity of the buffer was less than 5%, so that the solvent background in the concentrate was completely replaced by the acetate-sodium acetate buffer system (this process removes most of the free cofactors). Finally, the protein concentration was determined by the Coomassie brilliant blue method, and the protein concentration was adjusted to 10 mg / mL using the above buffer. SDS-PAGE electrophoresis and sequence analysis confirmed that the molecular weight of the target enzyme protein was approximately 54 kDa, yielding F428D mutant enzyme concentrate A.

[0026] Preparation Example 2: This preparation example provides a method for preparing a concentrated solution of wild-type deoxynivalenol degrading enzyme from Fusarium deoxynivalensis in an acetate buffer system, comprising the following steps: 1. Using the wild-type recombinant dehydrogenase gene from *Youhaiella tibetensis* as a template (sequence shown in SEQ ID NO. 1), without site-directed mutagenesis, the gene fragment was directly ligated into the *Pichia pastoris* expression vector pPICZαA, electroporated into *Pichia pastoris* X-33 competent cells, and wild-type recombinant strain Re-DON-WT was obtained by bleomycin plate screening.

[0027] 2. The recombinant strain was inoculated into a basic salt medium for fed-batch fermentation. The fermentation temperature was controlled at 30 degrees Celsius, the pH value was 6.0, and the dissolved oxygen was maintained above 30%. When the wet weight of the cells reached 200 g / L, methanol was fed to induce expression. During the induction period, pyrroloquinoline quinone disodium salt solution was added to the fermentation broth to maintain the final concentration of pyrroloquinoline quinone disodium salt in the fermentation broth at 30 μmol / L. The induction fermentation time was 80 hours.

[0028] 3. After fermentation, the cell bodies were removed by centrifugation, and the fermentation supernatant was collected. Tangential flow ultrafiltration was performed using a polyethersulfone membrane with a molecular weight cutoff of 10 kDa to concentrate the supernatant to one-tenth of its original volume. Subsequently, the concentrate was subjected to constant volume dialysis and filtration using a 50 mmol / L acetate-sodium acetate buffer (pH 6.0) until the difference between the conductivity of the permeate and the conductivity of the buffer was less than 5%, thus completely replacing the solvent background in the concentrate with the acetate-sodium acetate buffer system. Finally, the protein concentration was determined by the Coomassie brilliant blue method, and the protein concentration was adjusted to 10 mg / mL using the above buffer. The molecular weight of the wild-type enzyme protein was approximately 54 kDa, yielding wild-type enzyme concentrate B.

[0029] Preparation Example 3: This preparation example provides a method for preparing a concentrated solution of F428D mutant deoxynivalenol degrading enzyme in a phosphate-citrate buffer system, comprising the following steps: 1. Using the wild-type recombinant dehydrogenase gene from *Youhaiella tibetensis* as a template (sequence shown in SEQ ID NO. 1), site-directed mutagenesis was performed using overlap extension polymerase chain reaction (ELISA) to mutate the amino acid residue at position 428 by polar amino acid mutation, resulting in a mutant gene fragment. This mutant gene fragment was ligated into the *Pichia pastoris* expression vector pPICZαA, electroporated, and introduced into *Pichia pastoris* X-33 competent cells. The high-copy recombinant strain Re-DON-F428D was obtained by bleomycin plate selection.

[0030] 2. The recombinant strain was inoculated into a basic salt medium for fed-batch fermentation. The fermentation temperature was controlled at 30 degrees Celsius, the pH value was 6.0, and the dissolved oxygen was maintained above 30%. When the wet weight of the cells reached 200 g / L, methanol was fed to induce expression. During the induction period, pyrroloquinoline quinone disodium salt solution was added to the fermentation broth to maintain the final concentration of pyrroloquinoline quinone disodium salt in the fermentation broth at 30 μmol / L. The induction fermentation time was 80 hours.

[0031] 3. After fermentation, the cell bodies were removed by centrifugation, and the fermentation supernatant was collected. The supernatant was then concentrated by tangential flow ultrafiltration using a polyethersulfone membrane with a molecular weight cutoff of 10 kDa, reducing the volume to one-tenth of the original volume. Subsequently, the concentrate was subjected to constant-volume dialysis and filtration using a 20 mmol / L disodium hydrogen phosphate-citrate buffer (pH 6.0) until the difference between the conductivity of the permeate and the conductivity of the buffer was less than 5%, ensuring that the solvent background in the concentrate was completely replaced by the disodium hydrogen phosphate-citrate buffer system. Finally, the protein concentration was determined by the Coomassie brilliant blue method, and the protein concentration was adjusted to 10 mg / mL using the above buffer to obtain the control enzyme concentrate C. Example 1:

[0032] This embodiment provides a preparation process for a highly active deoxynivalenol-degrading enzyme from Fusarium nivale, comprising the following steps: S1. Take 1 liter of the concentrated F428D mutant enzyme solution A obtained in Preparation Example 1 and place it in a jacketed stirred reactor. Turn on the coolant to lower the temperature to 4 degrees Celsius. Under the condition of stirring speed of 150 rpm, slowly add 1.0 mol / L of zinc acetate dihydrate aqueous solution. According to the molecular weight of the enzyme protein, control the molar ratio of zinc acetate to enzyme protein to be 30:1. After the addition is completed, maintain a constant temperature of 4 degrees Celsius and stir at low speed for 3 hours. Utilize the weak coordination characteristics of acetate to promote the zinc ion to preferentially form stable coordination bonds with the F428D site and adjacent amino acids on the enzyme surface. S2. After the reaction is complete, keep stirring and slowly add 50g of trehalose (5% of the enzyme solution mass), 150g of maltodextrin (15% of the enzyme solution mass), and 0.1g of disodium pyrroloquinoline quinone to the reaction vessel; continue stirring for 30 minutes until all excipients are completely dissolved to obtain a homogeneous enzyme solution to be dried. S3. Pump the enzyme solution to be dried into a centrifugal spray drying tower for drying, control the inlet air temperature to be 150 degrees Celsius, the outlet air temperature to be 70 degrees Celsius, and the atomization pressure to be 0.3 MPa; collect the dry powder in the cyclone separator at the bottom of the tower to obtain the target enzyme preparation product. Implementation 2:

[0033] This embodiment provides a preparation process for a highly active deoxynivalenol-degrading enzyme from Fusarium nivale, comprising the following steps: S1. Take 1 liter of the concentrated F428D mutant enzyme solution A obtained in Preparation Example 1 (acetic acid-sodium acetate buffer system, protein concentration 10 mg / mL), place it in a jacketed stirred reactor, and cool it to 6 degrees Celsius. Under the condition of stirring speed of 120 rpm, slowly add 1.0 mol / L calcium acetate monohydrate aqueous solution. According to the molecular weight of the enzyme protein, control the molar ratio of added calcium acetate to enzyme protein to be 40:1. After the addition is completed, maintain a constant temperature of 6 degrees Celsius and stir for 2 hours to allow calcium ions to complete coordination and anchoring with the F428D site on the enzyme surface. S2. After the reaction is complete, add 30 g of trehalose (3% of the enzyme solution mass), 100 g of maltodextrin (10% of the enzyme solution mass), and 0.1 g of disodium pyrroloquinoline quinone to the enzyme solution; continue stirring until the excipients are completely dissolved to obtain the enzyme solution to be dried. S3. Pump the enzyme solution to be dried into a spray drying tower for drying, control the inlet air temperature to be 145 degrees Celsius, the outlet air temperature to be 65 degrees Celsius, and the atomization pressure to be 0.25 MPa; collect the dry powder at the bottom of the tower to obtain the target enzyme preparation product. Example 3:

[0034] This embodiment provides a preparation process for a highly active deoxynivalenol-degrading enzyme from Fusarium nivale, comprising the following steps: S1. Take 1 liter of the concentrated F428D mutant enzyme solution A obtained in Preparation Example 1 (acetic acid-sodium acetate buffer system, protein concentration 10 mg / mL), place it in a jacketed stirred reactor, and cool it to 10 degrees Celsius. Under the condition of stirring speed of 200 rpm, slowly add a mixed metal salt solution, which is composed of 1.0 mol / L zinc acetate dihydrate solution and 1.0 mol / L calcium acetate monohydrate solution at a volume ratio of 5:1. According to the molecular weight of the enzyme protein, control the molar ratio of the total metal ions added to the enzyme protein to be 20:1. After the addition is completed, maintain a constant temperature of 10 degrees Celsius and stir for 4 hours. S2. After the reaction is complete, add 80 g of trehalose (8% of the enzyme solution mass), 200 g of maltodextrin (20% of the enzyme solution mass), and 0.1 g of disodium pyrroloquinoline quinone to the enzyme solution; continue stirring until the excipients are completely dissolved to obtain the enzyme solution to be dried. S3. Pump the enzyme solution to be dried into a spray drying tower for drying, control the inlet air temperature to be 140 degrees Celsius, the outlet air temperature to be 65 degrees Celsius, and the atomization pressure to be 0.2 MPa; collect the dry powder at the bottom of the tower to obtain the target enzyme preparation product. Example 4:

[0035] This embodiment provides a preparation process for a highly active deoxynivalenol-degrading enzyme from Fusarium nivale, comprising the following steps: S1. Take 1 liter of the concentrated F428D mutant enzyme solution A obtained in Preparation Example 1 (acetic acid-sodium acetate buffer system, protein concentration 10 mg / mL), place it in a jacketed stirred reactor, and cool it to 8 degrees Celsius; under the condition of stirring speed of 180 rpm, slowly add 1.0 mol / L zinc acetate dihydrate aqueous solution; according to the molecular weight of the enzyme protein, control the molar ratio of zinc acetate to enzyme protein to be 60:1; after the addition is completed, maintain a constant temperature of 8 degrees Celsius and stir for 2.5 hours. S2. After the reaction is complete, add 60 g of trehalose (6% of the enzyme solution mass), 180 g of maltodextrin (18% of the enzyme solution mass), and 0.1 g of disodium pyrroloquinoline quinone to the enzyme solution; continue stirring until the excipients are completely dissolved to obtain the enzyme solution to be dried. S3. Pump the enzyme solution to be dried into a spray drying tower for drying, control the inlet air temperature to be 160 degrees Celsius, the outlet air temperature to be 75 degrees Celsius, and the atomization pressure to be 0.4 MPa; collect the dry powder at the bottom of the tower to obtain the target enzyme preparation product.

[0036] Comparative Example 1: Compared with Example 1, the difference is that zinc acetate solution is not added in step S1, but an equal volume of deionized water is added instead, and a metal coordination locking structure is not constructed. The remaining steps and parameters are the same.

[0037] This comparative example is used to verify the necessity of metal ion locking for improving heat resistance, that is, a stable structure cannot be formed when there are only mutation sites without metal binding.

[0038] Comparative Example 2: Compared with Example 1, the difference is that the raw material used in step S1 is the wild-type enzyme concentrate B obtained in Preparation Example 2, while the other steps and parameters are the same.

[0039] This comparative example is used to verify the specificity of the F428D mutation site as a charge anchor, that is, the wild-type enzyme cannot effectively bind metal ions under the same process, and the improvement in heat resistance is not significant.

[0040] Comparative Example 3: Compared with Example 1, the difference is that the raw material used in step S1 is the control enzyme concentrate C obtained in the phosphate-citric acid buffer system of Example 3, while the other steps and parameters are the same.

[0041] This comparative example is used to verify the key role of the acetate buffer system. Phosphate or citrate ions can precipitate or competitively chelate with zinc ions, causing metal ions to be unable to bind to enzyme proteins, and even producing turbidity, which can damage the stability of the process.

[0042] Comparative Example 4: Compared with Example 1, the difference is that the low-temperature constant temperature stirring reaction process is omitted in step S1. Instead, 1.0 mol / L of zinc acetate dihydrate aqueous solution is quickly added to the enzyme solution at room temperature, and steps S2 and S3 are performed immediately after mixing. The remaining steps and parameters are the same.

[0043] This comparative example is used to verify the necessity of the low-temperature slow coordination process parameters, that is, metal ions need specific thermodynamic time and kinetic processes to find and lock the F428D site, and instantaneous mixing cannot form a uniform coordination layer.

[0044] Test Example 1: This test case aims to verify the key role of the acetate buffer system constructed in the metal coordination process, and in particular to confirm whether the system can prevent non-specific precipitation of metal ions, thereby ensuring that metal ions are in a free state that can bind, and providing a material basis for subsequent enzyme surface coordination locking.

[0045] 1. Experimental Procedure 50 mL of reaction solution was taken from each of the coordination reaction vessels of Examples 1, 2, 3, and Comparative Example 3, before the addition of the protective agent and spray drying, after the reaction was completed. Comparative Example 2 was used as a control to eliminate the influence of the protein's properties on the solubility of the system.

[0046] After thoroughly vortexing and mixing each group of samples, immediately transfer them to a cuvette with an optical path of 1 cm. Measure the optical density (OD600) at 600 nm using a UV-Vis spectrophotometer. This value directly reflects the formation of insoluble particles or colloidal precipitates in the system; a lower value indicates a clearer system and better compatibility between the metal ions and the buffer solution.

[0047] The remaining sample was centrifuged at 12,000 rpm for 10 minutes to remove all insoluble precipitates. The supernatant was collected, and the concentration of metal ions (zinc or calcium) was determined using inductively coupled plasma atomic emission spectrometry. The retention rate of metal ions in the liquid phase was calculated based on the initial feed amount using the formula: Retention rate = (Metal concentration in supernatant / Theoretical feed concentration) × 100%. This indicator is used to quantify the proportion of metal ions that actually participate in or can participate in enzyme coordination.

[0048] 2. Experimental Data Table 1. Results of turbidity and metal ion retention rate in liquid phase for each reaction system.

[0049] 3. Conclusion Analysis According to Table 1 and Figure 1 Data analysis showed that Examples 1 to 3, using the acetate-sodium acetate buffer system, exhibited extremely low optical density values ​​(OD600 < 0.05) and near 100% metal ion liquid-phase retention. This indicates that in this weakly acidic carboxylate environment, both zinc and calcium ions, or mixtures of both, can exist stably in a dissolved state without any visible or instrumentally measurable precipitation reactions. Acetate, as a weak ligand, does not forcibly bind metal ions to form insoluble salts, thus ensuring that metal ions have thermodynamic freedom to bind to the enzyme protein surface.

[0050] In contrast, Comparative Example 3, using a traditional phosphate-citrate buffer system, exhibited extremely high turbidity (OD600 > 1.8), and the effective zinc ion content in the liquid phase was less than 9%. This directly confirms that in conventional biological buffers, phosphate or citrate ions can form sparingly soluble salts with extremely low solubility product constants with divalent metal ions, such as zinc phosphate or zinc citrate precipitates. This precipitation effect not only leads to the depletion of metal ions from the liquid phase, preventing them from participating in subsequent locking of the F428D site, but also the resulting fine particles become impurity nuclei during spray drying, disrupting the homogeneity of the enzyme preparation.

[0051] Although Comparative Example 2 is identical to Example 1 in physical properties, this only demonstrates the chemical compatibility of the acetate system and does not represent the achievement of enzyme stabilization. Combined with subsequent heat resistance test data, it can be inferred that the leap in heat resistance achieved in Example 1 is based on the chemical foundation that the acetate system prevents metal precipitation, allowing high concentrations of free metal ions to successfully attack and anchor the F428D mutation site. In contrast, Comparative Example 3, due to metal precipitation, could not achieve locking even with the mutation site, ultimately leading to process failure. This result strongly supports the inventive claim in this invention regarding the selection of the reaction medium as a key inventive point.

[0052] This test case quantitatively analyzes the residual metal ion content in the enzyme protein after dialysis treatment, aiming to verify the specific capture ability of the F428D mutation site for specific metal ions (such as zinc ions) and the strength of the enzyme-metal coordination bond formed therefrom.

[0053] Test Example 2: This test case quantitatively analyzes the residual metal ion content in the enzyme protein after rigorous dialysis treatment, aiming to verify the specific capture ability of the F428D mutation site for specific metal ions (such as zinc ions) and the strength of the enzyme-metal coordination bond formed therefrom.

[0054] 1. Experimental Procedure Weigh out 50 mg each of the spray-dried enzyme powder prepared in Example 1, Comparative Example 1, and Comparative Example 2. Add 10 mL of deionized water to completely reconstitute the powder, obtaining a homogeneous solution with a protein concentration of approximately 5 mg / mL. The reconstituted enzyme solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed in 1 liter of Tris-HCl buffer (50 mM, pH 7.0) free of any metal ions. Dialysis was performed at 4°C for 24 hours, with the dialysate replaced four times during this period (at 4, 8, 12, and 20 hours). This step utilizes a large concentration gradient to forcibly remove all free or weakly bound metal ions that are nonspecifically attached to the enzyme surface through weak interactions such as electrostatic adsorption and van der Waals forces, retaining only metal ions bound by strong coordination bonds.

[0055] After dialysis, the enzyme solution was removed from the bag. The actual protein concentration (mg / mL) of each sample was determined using a BCA protein quantification kit to correct for volume changes caused by dialysis. Then, 1 mL of enzyme solution was taken, 2 mL of concentrated nitric acid was added, and the mixture was heated in a digester at 120°C for 30 minutes until the solution was clear and transparent. Finally, the volume was adjusted to 10 mL with deionized water.

[0056] The total zinc content (µg / L) in the digest was determined using inductively coupled plasma mass spectrometry (ICP-MS). The number of moles of zinc ions bound to each enzyme molecule was calculated based on the molecular weight of the enzyme protein (approximately 54 kDa), serving as the core indicator for measuring coordination binding efficiency.

[0057] 2. Experimental Data Table 2. Results of determination of the molar binding ratio of enzyme protein to zinc ions after dialysis.

[0058] 3. Conclusion Analysis According to Table 2 and Figure 2 Analysis of experimental data showed that after 24 hours of intensive dialysis washing, the zinc / enzyme molar ratio of the F428D mutant enzyme in Example 1 remained stable at approximately 1.06. This value is very close to the theoretical stoichiometric ratio of 1:1, confirming that the aspartic acid at position 428 introduced in this invention successfully captured a zinc ion and formed a highly stable coordination complex in the suitable thermodynamic environment provided by acetate. This indicates the formation of a lock-in structure with high binding energy on the enzyme surface.

[0059] Although Comparative Example 2 also added sufficient zinc acetate during preparation, its final residual zinc / enzyme ratio was only 0.23 due to the lack of a carboxyl anchor at position 428. This residual zinc ions may be weakly adsorbed by non-specific sites on the enzyme surface (such as randomly distributed histidine residues or negatively charged surface regions), failing to form a specific spatially stable structure. This explains why the wild-type enzyme cannot achieve improved heat resistance even in the presence of metal. As for Comparative Example 1, its extremely low value (<0.05) is attributed to background noise from the instrument, confirming that the enzyme protein itself does not contain endogenous zinc.

[0060] In summary, this test case confirms the actual occurrence of the mechanism of mutation-induced anchoring and metal coordination locking at the molecular level. The F428D site acts as a high-affinity acceptor, while zinc acetate provides a usable ligand. This structural change is the fundamental reason for the subsequent improvement in macroscopic heat resistance.

[0061] Test Example 3: This test case aims to simulate the most demanding steam conditioning and pelleting processes in the feed industry. By applying instantaneous high temperature and high humidity stress to each group of enzyme preparations, the conformational retention ability and activity residue of the metal coordination-locked structure under extreme thermal conditions are examined.

[0062] 1. Experimental Procedure Weigh 1.0 g of each of the spray-dried enzyme powder prepared in Examples 1-4 and Comparative Examples 1-4, and place them in 10 mL heat-resistant stoppered glass test tubes. Add 9.0 mL of 50 mM acetate-sodium acetate buffer solution at pH 6.0, and vortex for 1 minute to fully dissolve the enzyme powder, thus preparing the test enzyme solution with consistent initial enzyme activity.

[0063] Turn on the constant temperature water bath and adjust the temperature to 85.0 degrees Celsius (±0.2 degrees Celsius). After the water temperature stabilizes, immerse each group of test tubes in the water bath simultaneously. The 85 degrees Celsius setting here corresponds to the steam conditioning temperature commonly used in feed mills.

[0064] At the three time points of 3 minutes, 5 minutes, and 10 minutes of heat treatment, the test tubes of the corresponding groups were quickly removed and immediately placed in an ice-water bath to cool for 5 minutes to terminate the heat denaturation process. Separately, unheated enzyme solutions from each group were used as controls (0 minutes, set as 100%).

[0065] Residual enzyme activity in each sample after cooling was determined by high performance liquid chromatography (HPLC). An appropriate amount of enzyme solution was reacted with the standard substrate deoxynivalenol at 37°C for 30 minutes, and the reaction was terminated by adding acetonitrile. Enzyme activity was calculated by detecting the reduction in substrate peak area.

[0066] The residual enzyme activity rate after heat treatment is calculated using the formula: Residual rate (%) = (Enzyme activity after heat treatment / Initial enzyme activity) × 100%. This indicator directly reflects the structural stability of enzyme molecules under high-temperature shock.

[0067] 2. Experimental Data Table 3. Residual activity data of enzyme preparations under 85℃ thermal shock conditions.

[0068] 3. Conclusion Analysis According to Table 3 and Figure 3 The thermal decay kinetics data shown reveal significant differences in heat resistance between the embodiments of this invention and the comparative examples. These data profoundly reveal the synergistic effect of molecular structure modification and process control from a macroscopic application perspective. After undergoing an extreme thermal shock of 85 degrees Celsius for up to 10 minutes, the enzyme preparations of Examples 1 to 4 maintained a high residual activity rate of 74% to 80%, demonstrating industrial thermal stability. This stability stems from the strong coordination network formed by the F428D mutation site and metal ions. This network locks the active site region of the enzyme, increasing the activation energy required for protein thermal denaturation, allowing it to maintain its correctly folded topology even under thermal perturbations.

[0069] In contrast, Comparative Examples 1 and 2 showed inactivation within 3 minutes of heat treatment, and almost all activity was lost (<8%) after 10 minutes. Introducing the F428D negative charge site alone without metal ion bridging, or adding metal ions alone without the crucial anchoring acceptor F428D, failed to construct an effective heat-resistant structure. In particular, data from Comparative Example 2 confirmed that the wild-type enzyme surface lacked specific strong binding sites, and free zinc ions could not play a role in stabilizing the backbone.

[0070] Although the chemical composition of Comparative Example 4 was completely identical to that of Example 1, the residual rate was only 35.6% and the standard deviation was large due to the omission of the low-temperature slow coordination step. This indicates that the metal ions seeking and locking onto specific sites on the enzyme surface is a thermodynamic process that requires overcoming steric hindrance. Instantaneous mixing leads to uneven metal coordination or loose coordination bonds. The low activity of Comparative Example 3 further confirms the conclusion of Test Example 1, namely that the metal precipitation caused by incompatible buffer systems directly deprives the enzyme molecules of their protective agents, causing its heat resistance to degrade to near the control group level. In summary, only when the three elements of specific mutation, correct solvent environment, and sufficient coordination reaction are simultaneously satisfied can a qualitative change in the heat resistance of enzyme preparations be achieved.

[0071] Test Example 4: This test case aims to evaluate the long-term stability of the F428D mutant enzyme preparation under simulated harsh storage conditions. By designing an accelerated aging experiment, the enzyme activity decay trend over the normal shelf life is predicted in a short period, thereby verifying whether the metal coordination structure can effectively inhibit spontaneous unfolding or conformational drift of the enzyme molecule over time.

[0072] 1. Experimental Procedure Freshly spray-dried enzyme powders prepared in Example 1, Comparative Example 1, and Comparative Example 4 were selected. Each group of samples was dispensed into multiple well-sealed aluminum foil bags, each bag was accurately weighed 5.0 grams, and heat-sealed to simulate finished product packaging.

[0073] The sealed sample was placed in a constant temperature and humidity incubator (LHS-150SC). The ambient temperature was set at 40.0 degrees Celsius (±0.5 degrees Celsius) and the relative humidity at 75% (±2%). These conditions comply with the accelerated stability testing standards specified in ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) Q1A(R2) guidance, and are used to simulate a high-temperature and high-humidity storage environment.

[0074] Sampling time points were set as day 0 (initial), day 15, day 30, day 45, and day 60. At each time point, three individually packaged sample bags were randomly selected from each group and allowed to return to room temperature.

[0075] Accurately weigh 0.1 g of enzyme powder, dissolve and dilute to volume with pH 6.0 buffer. Degrade its ability to the substrate deoxynivalenol was determined by HPLC. Using the measurement on day 0 as a baseline (100%), the relative enzyme activity residual rate at each time point was calculated.

[0076] 2. Experimental Data Table 4. Enzyme activity residual rate monitoring data under accelerated aging conditions at 40℃ / 75%RH

[0077] 3. Conclusion Analysis Based on the accelerated aging data recorded in Table 4 and Figure 4 The degradation curves presented show that after enduring a 60-day high-temperature and high-humidity (40℃ / 75%RH) challenge, the enzyme activity residue of the sample in Example 1 remained at a high level of 91.2%, and the overall degradation curve was smooth, demonstrating extremely strong anti-aging properties. Converted to room temperature (25℃), this data, according to the Arrhenius equation, indicates that the product has a stable shelf life of at least 18 to 24 months at room temperature. In contrast, the activity of Comparative Example 1, lacking metal protection, dropped below 70% by day 30 and remained at only 38.86% by day 60, exhibiting typical exponential inactivation characteristics.

[0078] The rapid inactivation in Comparative Example 1 reveals the thermodynamic instability of the recombinant protein. Without external restraint, the flexible regions within the protein molecule undergo random conformational respiration due to increased thermal motion; this cumulative micro-deformation eventually leads to the collapse of the active site. In Example 1, the coordination bonds formed between zinc ions and the F428D site and adjacent residues effectively reduce the conformational entropy of the molecular surface. This ion-locked structure not only resists instantaneous high-temperature shocks (as shown in Test Example 3) but also effectively inhibits slow denaturation under long-term thermal perturbation, maintaining the enzyme molecule in a tightly folded state with catalytic activity.

[0079] Although Comparative Example 4 had the same formulation as Example 1, its residual rate after 60 days (64.15%) was significantly lower than that of Example 1 due to the use of a transient mixing process, and the data dispersion was also larger. This further confirms the necessity of the low-temperature slow coordination process emphasized in this invention. The coordination of metal ions with the enzyme surface is a process that seeks the lowest energy state. Transient mixing may lead to the formation of metastable, weakly bound coordination structures. Such imperfect structures are prone to dissociation under long-term thermal stress, resulting in protective failure. Therefore, only through a finely controlled reaction kinetic process can a truly storage-resistant metal-enzyme supramolecular complex be constructed.

[0080] Test Example 5: This test case aims to investigate whether the metal coordination locking process negatively inhibits the catalytic activity of the enzyme. Although metal ions enhance structural rigidity, they may also hinder substrate access to the active site or limit the conformational changes required for catalysis. Therefore, this experiment measured the substrate degradation rate at different time points and plotted reaction kinetic curves to verify whether the enzyme preparation prepared in this invention maintains high heat resistance while still possessing efficient toxin scavenging ability.

[0081] 1. Experimental Procedure A phosphate-buffered saline (PBS, pH 7.0) containing 10 μg / mL deoxynivalenol standard was prepared as the base reaction medium to simulate toxin contamination. This concentration covers the level of severe contamination commonly found in the feed industry.

[0082] Accurately weigh the enzyme powders from Example 1, Example 3, Comparative Example 1, and Comparative Example 2, respectively. Reconstitute and dilute with deionized water to ensure that the final protein concentration in each reaction system is strictly controlled at 0.5 μg / mL.

[0083] To prevent errors in enzyme activity measurement caused by the loss of cofactors during the initial purification process and to ensure accurate characterization of the catalytic performance of the protein backbone itself, calcium chloride (CaCl2) and pyrroloquinoline quinone (PQQ) with a final concentration of 1.0 mmol / L and 20 μmol / L were added to the above reaction system.

[0084] After preheating the substrate solution in a constant temperature water bath at 37 degrees Celsius, the enzyme solution was added to initiate the reaction. At 5, 15, 30, and 60 minutes of reaction time, 1.0 mL of the reaction solution was taken from each reaction system.

[0085] Immediately add 1.0 mL of chromatographic grade methanol to the extracted reaction solution and vortex vigorously for 30 seconds to denature and precipitate the enzyme protein, thus terminating the catalytic reaction. Filter the mixture through a 0.22 μm microporous membrane and inject it into a high-performance liquid chromatograph for detection. The mobile phase was acetonitrile:water (20:80), the flow rate was 1.0 mL / min, and the detection wavelength was 218 nm. The residual toxin concentration at each time point was calculated based on the DON standard curve and converted to the cumulative degradation rate (%). Degradation rate = [(initial concentration - residual concentration) / initial concentration] × 100%.

[0086] 2. Experimental Data Table 5. Degradation kinetics data of different enzyme preparations for DON toxin

[0087] 3. Conclusion Analysis Based on the dynamic monitoring data in Table 5 and Figure 5 Based on comprehensive analysis, the metal coordination locking structure constructed in this invention not only did not cause significant damage to the catalytic activity of the enzyme, but also improved the substrate conversion efficiency under specific formulations.

[0088] Data shows that Example 3 exhibited the highest degradation rate at all time points, reaching 96.8% at 30 minutes, and its specific activity was significantly higher than other groups. This phenomenon suggests that the introduction of calcium ions may have a dual role: on the one hand, it synergistically reinforces the enzyme's external framework with zinc ions; on the other hand, it may act as a cofactor in charge transfer or substrate localization near the active site, thereby lowering the reaction energy barrier. In Example 1, the degradation rate reached 92.5% at 30 minutes. Compared with Comparative Example 1 without metal, the activity not only did not decrease due to structural rigidity but was actually slightly improved. This refutes the traditional view that improved stability necessarily sacrifices activity. It is speculated that the anchoring effect of metal ions makes the geometry of the active site pocket more regular, reducing ineffective conformational oscillations and increasing the probability of enzyme-substrate collision.

[0089] Conversely, Comparative Example 2 exhibited the worst catalytic performance, with a degradation rate of only 81.6% after 60 minutes, significantly lagging behind the other groups. This may be because the wild-type enzyme lacks a specific binding site at position 428, causing free zinc ions in solution to bind non-specifically to other regions on the enzyme surface (such as the entrance to the catalytic active site), resulting in non-competitive inhibition or steric hindrance, which prevents the substrate DON from entering the active pocket.

[0090] In summary, the F428D mutation and metal coordination process of this invention achieves a perfect balance between heat resistance and catalytic activity. This technical solution, while extending the survival time of the enzyme preparation in the high-temperature environment of feed processing through ion locking, fully preserves and even optimizes its toxin degradation ability in the physiological environment of the animal digestive tract (37°C), demonstrating that the enzyme preparation possesses extremely high industrial application value and biological efficacy.

[0091] 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 preparation process for a highly active deoxynivalenol-degrading enzyme from Fusarium nivale, characterized in that, Includes the following steps: S1. Take the concentrated solution of F428D mutant deoxynivalenol degrading enzyme in an acetate buffer system, and add an aqueous solution of metal acetate dropwise under stirring to carry out a coordination reaction; the metal acetate is selected from any one of zinc acetate and calcium acetate or a mixture thereof. S2. Add trehalose, maltodextrin and disodium pyrroloquinoline quinone to the enzyme solution after the reaction in step S1, stir to dissolve, and obtain the enzyme solution to be dried. S3. Spray dry the enzyme solution to be dried, collect the dry powder, and the target enzyme preparation product is obtained.

2. The preparation process of a highly active deoxynivalenol-degrading enzyme from Fusarium oxysporum according to claim 1, characterized in that, The temperature of the coordination reaction in step S1 is controlled between 4 and 10 degrees Celsius; the reaction time is between 2 and 4 hours; and the molar ratio of the metal ions in the metal acetate salt to the enzyme protein in the enzyme concentrate is between 20:1 and 60:

1.

3. The preparation process of a highly active deoxynivalenol-degrading enzyme from Fusarium oxysporum according to claim 1, characterized in that, The amount of trehalose added in step S2 is 3% to 8% of the enzyme solution mass in step S1, and the amount of maltodextrin added is 10% to 20% of the enzyme solution mass in step S1.

4. The preparation process of a highly active deoxynivalenol-degrading enzyme from Fusarium oxysporum according to claim 1, characterized in that, The control parameters for spray drying in step S3 are: inlet air temperature of 140°C to 160°C, outlet air temperature of 65°C to 75°C, and atomization pressure of 0.2 MPa to 0.4 MPa.

5. The preparation process of a highly active deoxynivalenol-degrading enzyme from Fusarium oxysporum according to claim 1, characterized in that, The preparation method of the F428D mutant deoxynivalenol degrading enzyme concentrate in the acetate buffer system described in step S1 includes: collecting the fermentation supernatant containing the target enzyme, concentrating it by tangential flow ultrafiltration using a membrane with a molecular weight cutoff of 10 kDalton, and then dialyzing the concentrate with a constant volume using an acetate-sodium acetate buffer solution of 50 mmol / L until the difference between the conductivity of the permeate and the conductivity of the buffer solution is less than 5%.

6. The preparation process of a highly active deoxynivalenol-degrading enzyme from Fusarium oxysporum according to claim 1, characterized in that, The pH of the acetate-sodium acetate buffer solution is 6.0; the protein concentration in the F428D mutant deoxynivalenol degrading enzyme concentrate is adjusted to 10 mg / mL.

7. The preparation process of a highly active deoxynivalenol-degrading enzyme from Fusarium oxysporum according to claim 6, characterized in that, The amino acid sequence of the F428D mutant deoxynivalenol degrading enzyme is shown in SEQ ID NO.4, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.

3.

8. The preparation process of a highly active deoxynivalenol-degrading enzyme from Fusarium oxysporum according to claim 6, characterized in that, The F428D mutant deoxynivalenol degrading enzyme concentrate was obtained by fermenting recombinant Pichia pastoris, which contains an expression vector constructed by linking the gene fragment shown in SEQ ID NO. 3 to the pPICZαA vector and introducing it into Pichia pastoris X-33 competent cells.

9. The preparation process of a highly active deoxynivalenol-degrading enzyme from Fusarium oxysporum according to claim 8, characterized in that, The fermentation process includes: adding methanol to induce expression when the wet weight of the cells reaches 200 g / L, and adding a solution of disodium pyrroloquinoline quinone during the induction period to maintain the final concentration of disodium pyrroloquinoline quinone in the fermentation broth at 30 μmol / L.

10. The preparation process of a highly active deoxynivalenol-degrading enzyme from Fusarium oxysporum according to claim 1, characterized in that, The aqueous solution of the metal acetate in step S1 is a 1.0 mol / L zinc acetate dihydrate or a 1.0 mol / L calcium acetate monohydrate; the molecular weight of the enzyme protein in the enzyme concentrate is 54 kDaltons.

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