Composite protective agent for liquid laccase and application thereof

Abstract

The invention discloses a composite protective agent for liquid laccase and application of the composite protective agent, and belongs to the technical field of bioengineering. The protective agent takes a citric acid-disodium hydrogen phosphate buffer solution with the pH value of 4.5-6.5 as a solvent, and comprises propylene glycol, glycerol, sorbitol, cane sugar, maltodextrin, glycine, trehalose, potassium sorbate, diacetic acid and a mediator. The components form a stable microenvironment around enzyme molecules through a synergistic effect, the polyhydric alcohols and the saccharides stabilize an enzyme structure through hydrogen bonds and glassy state protection, the glycine plays a buffering role, and the mediator has dual functions of oxidation resistance and mediated catalytic reaction. Experiments prove that the protective agent can remarkably improve the thermal stability and storage stability of the liquid laccase, the enzyme activity retention rate reaches 86.7% after the liquid laccase is treated at 50 DEG C for 24 h, and the enzyme activity retention rate is still kept at 85.3% after the liquid laccase is stored at 37 DEG C for 180 days. The laccase is especially suitable for degrading aflatoxin B1, has a degradation effect obviously better than that of unprotected laccase, and can be widely applied to the fields of food industry, feed industry and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a composite protective agent for liquid laccase and application thereof. BACKGROUND

[0002] Laccase (EC 1.10.3.2) is a copper-containing polyphenol oxidase widely existing in fungi, bacteria and plants, has the advantages of wide substrate, mild catalytic conditions, reaction product being water and the like, and is praised as a "green catalyst". Laccase shows broad application prospects in the fields of food processing, papermaking, textile, environmental pollutant degradation and mycotoxin detoxification.

[0003] Especially in the field of food safety, mycotoxin (such as aflatoxin AFB1, zearalenone ZEN and the like) pollution has become a global problem, and traditional physical and chemical detoxification methods have problems of low efficiency, great loss of nutrients, easy to cause secondary pollution and the like. Laccase is regarded as an ideal biological detoxification enzyme due to its efficient, specific and environmentally friendly catalytic characteristics. Studies have shown that laccase can effectively degrade a variety of mycotoxins such as AFB1 under appropriate conditions, and the degradation rate can be more than 80%, or even close to complete degradation (such as in the presence of some mediators).

[0004] However, laccase faces many challenges in practical application: 1. Poor stability: free laccase is sensitive to environmental factors such as temperature, pH, ionic strength and inhibitors, and is easy to inactivate; 2. Difficult storage and transportation: liquid laccase is prone to enzymatic activity decline during storage, and although freeze-drying treatment can prolong the shelf life, the activity loss after reconstitution is significant (such as literature reports that the AFB1 degradation rate decreases by about 34% after freeze-drying); 3. Limited application scenarios: laccases from different sources have strong substrate specificity, and have low degradation efficiency for some toxins (such as azo dyes, ZEN and the like), and need to rely on mediator systems to play a high-efficiency degradation role; 4. Poor reusability: free enzymes are difficult to recycle, increasing the use cost.

[0005] In the prior art, although the immobilized enzyme technology improves the stability and reusability of the enzyme to some extent, there are still problems such as complex carrier selection, complicated preparation process, enzyme activity loss and the like. In addition, the existing protective agents are mostly for solid or freeze-dried enzyme preparations, and there are few studies on the long-term stability protection of liquid laccase. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a complex protective agent for liquid laccase and application thereof. The protective agent can provide a stable microenvironment for laccase molecules through synergistic effect of specific components, significantly slows down the attenuation of enzyme activity, and prolongs the storage stability. The application of the laccase can effectively degrade aflatoxin B1 and can be applied in the fields of food industry and feed industry.

[0007] To achieve the above object, the present application adopts the following technical solutions: In a first aspect, the present application provides a complex protective agent for liquid laccase, taking citric acid-disodium hydrogen phosphate buffer as a solvent, and comprising the following components: propylene glycol, glycerol, sorbitol, sucrose, malt dextrin, aminoacetic acid, trehalose, potassium sorbate, diacetic acid, and mediator.

[0008] The laccase solution described in the present application is derived from fungi, such as white-rot fungi, and is prepared through liquid submerged fermentation, extraction, and concentration processes.

[0009] The mediator in the present application includes one or a combination of 2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTH), 1-hydroxybenzotriazole (HBT), gallic acid, gallic acid ethyl ester, gallic acid propyl ester, ferulic acid, vanillin, and acetyl vanillin; preferably, the mediator is gallic acid propyl ester.

[0010] Preferably, the liquid laccase protective agent comprises the following components: 10-40% propylene glycol, 5-30% glycerol, 0-20% sorbitol, 0-10% sucrose, 0-10% malt dextrin, 2-8% aminoacetic acid, 3-10% trehalose, 0.05-2.0% potassium sorbate, 0.05-2.0% diacetic acid, and 0.01-0.5% mediator.

[0011] Preferably, the liquid laccase protective agent comprises the following components: 15% propylene glycol, 25% glycerol, 10% sorbitol, 5% sucrose, 2% malt dextrin, 2% aminoacetic acid, 5% trehalose, 0.05% potassium sorbate, 0.05% diacetic acid, and 0.05% gallic acid propyl ester.

[0012] In one or more examples, the propylene glycol, glycerol, sorbitol, sucrose, malt dextrin, aminoacetic acid, and trehalose are food grade.

[0013] Preferably, the pH of the citric acid-disodium hydrogen phosphate buffer is 4.5-6.5; preferably, the pH is 5.0.

[0014] Preferably, the concentration of the citric acid-disodium hydrogen phosphate buffer is 0.05-0.2M; preferably, the concentration is 0.1M.

[0015] Secondly, the present invention also provides a method for preparing the composite protective agent, comprising: dissolving enzyme solution, propylene glycol, glycerol, sorbitol, sucrose, maltodextrin, glycine, trehalose, potassium sorbate, diacetic acid, and mediator one by one in a citrate-disodium hydrogen phosphate buffer solution, mixing evenly, and then aseptically filtering and aseptically filling the solution.

[0016] Furthermore, this invention claims protection for the use of the aforementioned composite protective agent in improving the storage stability of liquid laccase.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Strong synergistic effect of the formulation: This protective agent is designed to meet the stability requirements of laccase, and the components work synergistically in citrate-disodium hydrogen phosphate buffer. Propylene glycol, glycerol, and sorbitol, as polyols, prevent denaturation by forming hydrogen bonds and maintaining the hydration layer of enzyme molecules; trehalose, sucrose, and dextrin can form a protective glassy structure on the surface of enzyme molecules; glycine, as a zwitterion, plays a buffering and stabilizing role; the mediator can act as an antioxidant, effectively preventing the oxidative inactivation of enzyme molecules, and can also preferentially oxidize laccase to generate highly active intermediates (such as free radicals), which then diffuse out to oxidize those large and complex substrates that laccase cannot directly contact or oxidize; 2. Significantly improved stability: Experiments have shown that liquid laccase with added protective agent of this invention, after being stored at 4℃ and 37℃ for 90 days, has a significantly higher enzyme activity retention rate than the control group without protective agent (for example, more than 60% higher at 37℃), effectively extending the product's shelf life. 3. Strong degradation effect on fungal toxins: Experiments have shown that liquid laccase with added protective agent of this invention can effectively degrade the fungal toxin aflatoxin B1. 4. Low application cost: The raw materials used in this protective agent are readily available and low in cost. The preparation process is simple and suitable for large-scale production, which can effectively reduce the storage and application costs of liquid laccase.

[0018] The working principle and beneficial effects of this invention are as follows: I. Synergistic effect of multiple stabilization mechanisms: 1. Propylene glycol, glycerol, and sorbitol: As polyol systems, they effectively reduce the water activity of the system by forming a hydrogen bond network and form a protective hydration layer around the enzyme molecule to prevent the enzyme from denaturing and becoming inactive at high or low temperatures. 2. Trehalose, sucrose, and dextrin: can form a glassy protective film on the surface of enzyme proteins, effectively fixing the native conformation of enzyme molecules and preventing them from unfolding and aggregating; 3. Glycine (aminoacetic acid): As an amphoteric amino acid, it can regulate the micro-polar environment of the solution, stabilize the charged state of the enzyme, and effectively buffer the impact of external pH fluctuations. 4. Mediator: As a highly efficient antioxidant, it can scavenge dissolved oxygen and free radicals, preventing the oxidation of copper ions at the active center of laccase and the oxidative degradation of the enzyme protein itself, thus maintaining long-term activity. Furthermore, the mediator acts as an "electron shuttle" in liquid laccase. In application, laccase first oxidizes the mediator, generating highly active mediator free radicals. These free radicals can diffuse to regions of fungal toxins (such as AFB1 and ZEN) that laccase molecules cannot directly access, attacking and destroying their toxic structures (such as coumarin lactone rings and lactone rings) through electron transfer, degrading them into low-toxicity or non-toxic small molecule products. This mechanism greatly expands the range of action and degradation efficiency of laccase, achieving highly efficient biological detoxification. 5. pH 4.5-6.5 buffer system: This pH value is close to the optimal pH range of this fungal laccase, providing an initial and stable microenvironment for the enzyme, reducing inactivation caused by pH incompatibility from the source; II. Significantly Improved Stability: 1. Thermal stability: Liquid laccase treated with this protectant retains more than 85% of its enzyme activity after being treated at 50°C for 24 hours, which is significantly better than unprotected laccase (usually less than 20%). 2. Storage stability: After being stored at room temperature and away from light for 90 days at 37℃, the enzyme activity retention rate can still be maintained at over 83%, which greatly facilitates transportation, storage and on-site application; III. Wide range of applications: Particularly suitable for the degradation of mycotoxins: This protectant system not only stabilizes laccase, but its mild environment and the contained mediator precursors (such as the degradation products of propyl gallate, which may have certain mediator functions) synergistically promote the degradation efficiency of laccase against mycotoxins such as aflatoxin (AFB1). Experiments show that laccase solutions using this protectant have a 25%-80% higher degradation rate than the control group using ordinary buffer solutions when treating mycotoxin-contaminated food or feed. IV. Diverse Application Scenarios: In addition to the degradation of fungal toxins, this liquid laccase preparation is also suitable for multiple fields such as textile bio-dyeing, pulp bio-bleaching, and dyeing wastewater treatment, demonstrating universal stability and catalytic efficiency.

[0019] In summary, this invention provides a comprehensive and efficient solution for stabilizing liquid laccase through the scientific ratio and synergistic effect of its components, thus solving a key bottleneck problem in the industrial application of liquid laccase preparations. Attached Figure Description

[0020] Figure 1 The results of the thermal stability test of the protective agent and the control sample of this invention at 50°C.

[0021] Figure 2 Comparison of the degradation effects of the protective agent of this invention and the control sample on aflatoxin B1. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0023] Example 1: Preparation of composite protective agent.

[0024] Prepare a 0.1 M citrate-disodium hydrogen phosphate buffer solution with a pH of 5.0 as the base solvent.

[0025] Add each food-grade component to the buffer solution in the following volume percentages, and stir thoroughly until completely dissolved: Propylene glycol: 15%; Glycerin: 25%; Sorbitol: 10%; Sucrose: 5%; Maltodextrin: 2%; Glycine: 2%; Trehalose: 5%; Potassium sorbate: 0.05%; Diacetic acid: 0.05%; Propyl gallate: 0.05%.

[0026] The final volume was brought up to 100% using the buffer solution from step 1, mixed thoroughly, and then filtered through a 0.22 μm filter membrane for sterilization to obtain the liquid laccase composite protectant described in this invention. It was then sealed and stored at 4°C for later use.

[0027] Example 2: Preparation of stabilized liquid laccase preparation.

[0028] Take the concentrated laccase solution (enzyme activity >20000 U / mL) from white-rot fungi and mix it with the composite protectant prepared in Example 1 at a volume ratio of 1:1 (or n) (to prepare a liquid enzyme preparation containing 10000 U / mL of laccase). Gently shake to mix, and a stable liquid laccase preparation (experimental group) is obtained. At the same time, a control group is set up: the same batch of concentrated laccase solution is diluted with an equal volume of pH 5.0 ordinary citrate-disodium hydrogen phosphate buffer without the protectant.

[0029] Example 3: Experiment on degradation of aflatoxin B1 (AFB1) by liquid laccase containing composite protective agent.

[0030] Aflatoxin B1 (AFB1) is the most potent and widely harmful aflatoxin, exhibiting high carcinogenicity and acute toxicity. This study investigated the optimal reaction conditions using a biomimetic enzyme catalyst to target AFB1. AFB1 standard was dissolved in methanol to prepare a stock solution with a concentration of 0.1 mg / mL. In degradation experiments, liquid laccase containing a complex protective agent was added to the aflatoxin-containing reaction mixture in 100 mM Tris-HCl buffer (pH 5.0) at 25°C. The final substrate concentration was 5 μg / mL, and the final laccase concentrations were 2000 U / L, 4000 U / L, 6000 U / L, 8000 U / L, and 10000 U / L. The reaction was terminated by adding three times the volume of methanol after 0 h, 2 h, 4 h, 6 h, and 8 h, respectively. After the reaction, the samples were analyzed by HPLC. HPLC detection was performed using a 5 μm amino acid column, a fluorescence detector, a column temperature of 30 °C, and a flow rate of 0.5 mL / min; the mobile phase was acetonitrile-water; the injection volume was 5 μL; the excitation wavelength was 360 nm and the emission wavelength was 440 nm.

[0031] The percentage of aflatoxin degradation was calculated using the following equation: Aflatoxin degradation rate (%) = [(C0 - Ct) / C0] × 100 In the formula: C0 represents the concentration of aflatoxin in the control group, and Ct represents the concentration of aflatoxin in the experimental group.

[0032] The results are as follows Figure 2 As shown, after reacting at 25°C for 8 hours, liquid laccase containing the composite protective agent was found to have a high AFB1 degradation rate.

[0033] Comparative Example 1: Stability Test The experimental and control samples prepared in Example 2 were stored at 4°C and 37°C, respectively, and their residual enzyme activity was measured periodically to assess storage stability. The results are shown in the table below: Results Analysis: Data show that the composite protectant of this invention can significantly improve the long-term storage stability of liquid laccase. After 180 days, the enzyme activity retention rate of the experimental group under both room temperature and refrigeration conditions was much higher than that of the control group, proving the effectiveness of the protectant of this invention.

[0034] Comparative Example 2: Thermal Stability Test The experimental and control samples were placed in a 50℃ water bath for accelerated aging experiments, and enzyme activity was monitored periodically. After 24 hours, the laccase activity retention rate of the experimental group was 86.7%, while that of the control group was only 18.5%. The results indicate that the protective agent of this invention can greatly enhance the thermal stability of laccase.

[0035] In summary, the liquid laccase composite protectant provided by this invention has a scientifically sound formula, is easy to prepare, and can significantly inhibit the decline in activity of liquid laccase during storage, thus extending its shelf life and showing good prospects for industrial application.

[0036] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite protective agent for liquid laccase, characterized in that, The solution is a citrate-disodium hydrogen phosphate buffer containing the following components by volume percentage: 10-40% propylene glycol, 5-30% glycerol, 0-20% sorbitol, 0-10% sucrose, 0-10% maltodextrin, 2-8% glycine, 3-10% trehalose, 0.05-2.0% potassium sorbate, 0.05-2.0% diacetic acid, and 0.01-0.5% mediator.

2. The composite protective agent according to claim 1, characterized in that, The mediator is selected from one or more combinations of 2,2'-adiazon-bis(3-ethylbenzothiazoline-6-sulfonic acid), 1-hydroxybenzotriazole, gallic acid, ethyl gallate, propyl gallate, ferulic acid, vanillin, and acetylsylsyringone.

3. The composite protective agent according to claim 2, characterized in that, The mediator is propyl gallate.

4. The composite protective agent according to claim 3, characterized in that, It contains the following components by volume percentage: 15% propylene glycol, 25% glycerol, 10% sorbitol, 5% sucrose, 2% maltodextrin, 2% glycine, 5% trehalose, 0.05% potassium sorbate, 0.05% diacetic acid, and 0.05% propyl gallate.

5. The composite protective agent according to claim 1, characterized in that, The citrate-disodium hydrogen phosphate buffer solution has a pH of 4.5-6.5 and a concentration of 0.05-0.2M.

6. A stabilized liquid laccase preparation, characterized in that, It is prepared by mixing the composite protective agent according to any one of claims 1-6 with laccase solution at a volume ratio of 1:1 to 1:

5.

7. The stabilized liquid laccase preparation according to claim 6, characterized in that, The laccase is derived from fungi.

8. The use of the composite protectant according to any one of claims 1-6 in the preparation of an enzyme preparation for degrading aflatoxin B1.

9. The use of the stabilized liquid laccase preparation according to claim 7 or 8 in the degradation of aflatoxin B1.

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