Use of a multicopper oxidase in the degradation of nucleic acids

Abstract

This invention relates to the field of molecular biology, specifically to the application of a multi-copper oxidase in the degradation of nucleic acids. Its core applications include two aspects: first, using the multi-copper oxidase as a soil nutrient amendment to activate soil phosphorus by enzymatically hydrolyzing nucleic acids and releasing available phosphorus; second, degrading free pathogenic nucleic acids in public and cold chain environments to block the environmental transmission of pathogens and prevent human infection. The multi-copper oxidase can be derived from pure cultures, with an enzyme activity of 100-1000 U·mL. ‑1 The optimal pH is 4.0–6.5, and the optimal temperature is 25–55℃. In soil applications, the aqueous solution of polycopper oxidase is used at a concentration of 0.1–5 U·g. ‑1 When applied to soil, the nucleic acid degradation rate is ≥90%, and the available phosphorus is increased by 20-50 mg·kg. ‑1 In environmental disinfection, 0.5–20 U / mL ‑1 The degradation efficiency of the multi-copper oxidase is ≥95% after 10-120 minutes of action, and remains ≥85% even under cold chain conditions. This invention expands the application scenarios of multi-copper oxidase, combining the advantages of environmental friendliness, high efficiency and stability, and solves the problems of soil degradation and pathogen transmission control.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to the application of a multi-copper oxidase in the degradation of nucleic acids. Background Technology

[0002] Currently, soil fertility presents a complex situation characterized by "moderate to low levels, significant regional differences, and prominent degradation problems." Overall, approximately 70% of arable land is classified as low- to medium-yield land, with low average soil organic matter content, even below 1% in some areas. Imbalances in major nutrients such as nitrogen, phosphorus, and potassium are also prevalent. Furthermore, the phosphorus contained in soils with high organic matter content cannot be directly utilized by plants.

[0003] Currently, chemical fertilizers remain the core means of quickly replenishing crop nutrients and ensuring short-term production capacity in grain production. However, they can only enhance "quick-acting nutrients" and cannot improve core fertility elements such as soil structure, organic matter, and biological activity. Long-term reliance on chemical fertilizers can also lead to soil compaction, acidification, and nutrient imbalance. In contrast, enzymatic methods, due to their high specificity and environmental friendliness, are gradually becoming an important and effective way to replenish soil fertility. Polycopper oxidase, a polyphenol oxidase containing copper ions, can effectively degrade nucleic acids in the soil, releasing inorganic phosphorus through enzymatic hydrolysis, thus replenishing soil fertility and supplying it to plants.

[0004] Environmental transmission to humans refers to the indirect transmission of bacteria and viruses from the environment to humans through contamination of surfaces. For example, viruses can survive for days to weeks in refrigerated / frozen environments during cold chain transportation; and for hours to days on hard surfaces such as stainless steel and plastic, thus causing infection. Enzymatic hydrolysis methods, with copper oxidases at their core, can effectively degrade harmful nucleic acids in the environment, preventing environmental transmission to humans.

[0005] In view of the above-mentioned defects, the inventors of this invention have finally obtained this invention after a long period of research and practice. Summary of the Invention

[0006] The purpose of this invention is to solve the problem of how to release inorganic phosphorus through the hydrolysis of copper oxidase, replenish soil fertility, supply plants, and effectively degrade harmful nucleic acids in the environment, thereby avoiding environmental transmission to humans. This invention provides an application of copper oxidase in the degradation of nucleic acids.

[0007] To achieve the above objectives, this invention discloses the application of a multi-copper oxidase in the degradation of nucleic acids. The multi-copper oxidase, as a soil nutrient amendment, releases available phosphorus through enzymatic hydrolysis of nucleic acids, thereby activating soil phosphorus.

[0008] The copper oxidase is laccase or manganese peroxidase, the amino acid sequence of which is shown in SEQ ID NO.1 and the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The specific application methods are as follows:

[0010] S1, Prepare an aqueous solution of copper oxidase by adding a nonionic surfactant as a dispersant;

[0011] S2, spray the copper oxidase aqueous solution evenly onto the surface of the soil to be treated or mix it evenly with the topsoil of the soil to be treated to carry out the reaction.

[0012] In step S1, the polycopper oxidase is a purified culture-derived polycopper oxidase with an enzyme activity of 100-1000 U·mL. -1 The culture conditions for polycopper oxidase were as follows: pH of the culture medium was 4.0–6.5, and the culture temperature was 25–55℃. The amount of nonionic surfactant added was 0.01–0.1% of the total volume of the polycopper oxidase aqueous solution. The nonionic surfactant was TX-100.

[0013] In step S2, the reaction conditions are: soil temperature of 15-40℃, water content of 60-80% of field capacity, and reaction time of 24-72 hours.

[0014] In step S2, the amount of the polycopper oxidase applied is 0.1~5 U·g. -1 soil.

[0015] The present invention also discloses the application of a multi-copper oxidase in the degradation of nucleic acids. The multi-copper oxidase blocks the environmental transmission of pathogens mediated by the degradation of free nucleic acids in public environments and cold chain transportation environments, thereby preventing human infection. The multi-copper oxidase is laccase or manganese peroxidase. The amino acid sequence of the laccase is shown in SEQ ID NO.1, and the amino acid sequence of the manganese peroxidase is shown in SEQ ID NO.2.

[0016] The specific application methods are as follows:

[0017] A1. Prepare a copper oxidase working solution and apply the working solution to the target environment surface or space using atomized spray, wiping or slow-release device to ensure coverage of all areas where free nucleic acids may exist.

[0018] A2, after the action is complete, remove the residue by rinsing with sterile water or air drying.

[0019] In step A1, when preparing the multi-copper oxidase working solution, 0.1-1% of lignin derivatives are added as a mediator, based on the total volume of the multi-copper oxidase working solution. The multi-copper oxidase is a recombinant multi-copper oxidase or a modified product of a natural multi-copper oxidase. The modification includes, but is not limited to, PEG modification, glycosylation modification, or immobilization modification.

[0020] In step A2, the concentration of the polycopper oxidase is 0.5~20 U·mL. -1 ,

[0021] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention is the first to apply multi-copper oxidase degradation of nucleic acids to the interdisciplinary field of agriculture, cold chain logistics safety, and molecular biology. In the agricultural field, it is the first time that multi-copper oxidase has been used to supplement soil fertility. Furthermore, this method can effectively degrade nucleic acids in the soil, releasing inorganic phosphorus through enzymatic hydrolysis, avoiding soil compaction, acidification, and nutrient imbalance caused by conventional fertilization. In the interdisciplinary field of cold chain logistics safety and molecular biology, it innovatively applies multi-copper oxidase to enzymatic degradation of environmental nucleic acids. This innovation provides another new option for enzymatic degradation of environmental nucleic acids. Attached Figure Description

[0022] Figure 1 Bar chart (a) and line graph (b) showing the effects of phosphorus stress on polycopper oxidase and Escherichia coli;

[0023] Figure 2 plasmid (5 ng·µL) -1 Electrophoresis images after reaction with low-dose multi-copper oxidase (N: control; OC: open circular DNA; Lin: linear DNA; SC: supercoiled DNA; M: Trans2K Plus DNA Marker) Lanes 1-4: Samples reacted with 0.05, 0.1, 0.25, and 0.5 U·mL⁻¹, respectively. -1 Incubate with copper oxidase for 10 min; lanes 5-8: samples were incubated with 0.05, 0.1, 0.25, and 0.5 U·mL⁻¹, respectively. -1 Incubate with copper oxidase for 30 min;

[0024] Figure 3 Electrophoretic patterns of plasmid and cDNA after treatment with copper oxidase. (a) Plasmid; (b) cDNA. Note: N is the control group; OC is open circular DNA; Lin is linear DNA; SC is supercoiled DNA; M is the 2K Plus DNA molecular weight standard. (b) Lanes 1-5: Samples were mixed with 1, 2, 4, 8, and 16 U·mL, respectively. -1 Incubate with copper oxidase for 30 min; in lanes 6-10: samples are mixed with 16 U / mL of the enzyme. -1Multi-copper oxidase was incubated for 60, 90, 120, 150, and 180 min;

[0025] Figure 4 Electrophoretic patterns of plasmids and cDNA after water treatment. (a) Lane 1: Plasmid; Lane 2: Plasmid digested overnight with Hind III restriction enzyme; Lanes 3-10: Negative control samples of plasmid treated with double-distilled water for 1-8 h. (b) Lane 1: cDNA; Lane 2: cDNA treated with inactivating enzyme; Lanes 3-7: cDNA samples treated with double-distilled water for 30, 60, 90, 120, 150, and 180 min. Note: N is the control group; OC is open circular DNA; Lin is linear DNA; SC is supercoiled DNA; M is the 2K Plus DNA molecular weight standard.

[0026] Figure 5 These are atomic force microscopy (AFM) images of cDNA and plasmid after treatment with copper oxidase. (a, b, c) are AFM images of copper oxidase, plasmid, and cDNA, respectively; (d, e, f) are corresponding images of... Figure 3 Lane 1, Figure 3 Lane 5 and Figure 3 DNA atomic force microscopy image of sample b, lane 5. Note: AFM is an abbreviation for Atomic Force Microscopy. Detailed Implementation

[0027] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0028] Example 1

[0029] Copper oxidases degrade nucleic acids to supply phosphorus:

[0030] In this embodiment, the copper oxidase is laccase, and the amino acid sequence of laccase is shown below:

[0031] (SEQ ID NO.1).

[0032] I. Materials and Methods

[0033] 1. Culture medium preparation

[0034] LB medium (in 1 L): 10 g Bacto tryptone, 5 g Bacto yeast extract, 10 g NaCl, pH 7.0.

[0035] MOPS medium (per L): glucose 10 g, NH4Cl 0.51 g, MgCl2 0.107 g, K2SO4 48 mg, FeSO4·7H2O 2.8 mg, CaCl2·2H2O 0.0735 mg, NaCl 29.2 g, plus 20 μL of trace element solution (containing H3BO3 1.24 g, CoCl2 0.36 g, MnCl2 0.8 g, (NH4)6Mo7O3 per L). 24 ·4H2O 0.18 g, CuSO4 0.12g, ZnSO4 0.14 g).

[0036] Positive control medium (PC): MOPS + K2HPO4 2.3 g·L -1 ;

[0037] Nucleic acid culture medium (NA): MOPS + herring sperm DNA 1 mg / mL -1 .

[0038] Lac MOPS low-phosphorus medium containing laccase (per L): glucose 10 g, NH4Cl 0.51 g, MgCl2 0.107 g, K2SO4 48 mg, FeSO4·7H2O 2.8 mg, CaCl2·2H2O 0.0735 mg, NaCl 29.2 g, plus 20 μL of trace element solution (containing H3BO3 1.24 g, CoCl2 0.36 g, MnCl2 0.8 g, (NH4)6Mo7O3 per L). 24 ·4H2 O 0.18 g, CuSO4 0.12 g, ZnSO4 0.14 g), 0.5 U·mL -1 Laccase.

[0039] Lac NA (laccase-containing nucleic acid medium): MOPS + herring sperm DNA 1 mg / mL -1 0.5 U·mL -1 Laccase;

[0040] The pH of each culture medium was adjusted to 7.2 before sterilization, and laccase was added after being sterilized by a 0.22 μm filter membrane.

[0041] 2. E. coli survival rate test

[0042] Escherichia coli (DH5α) strain preserved at -80℃ in glycerol was revived in LB broth and plated onto 1.5% solid LB agar plates. Single colonies were picked and inoculated into 50 mL MOPS, PC, NA, Lac MOPS, and Lac NA media (500 mL shake flasks), respectively, and incubated at 37℃ with shaking at 280 rpm. Samples were taken at regular intervals to measure OD. 600 To compare changes in bacterial cell concentration under different phosphorus conditions and in the presence of laccase, and to evaluate the effect of laccase on the survival and growth of Escherichia coli.

[0043] II. Results and Analysis

[0044] Due to the prevalence of phosphorus stress, this study cultured E. coli DH5α (a mutagenic strain with immunodeficiency to exogenous DNA) to investigate the relationship between laccase-mediated DNA degradation and phosphorus supply in this strain. Figure 1 As shown in Figure a, during the 24–30 hours of cultivation, the maximum bacterial concentration in the laccase-containing medium was 3.8 times that in the non-laccase-containing medium; by 30 hours, this concentration difference had narrowed to 1.3 times. Although the bacterial growth rate in the fermentation medium was very slow, significant growth was still observed. We hypothesize that bacteria possess an inducible regulatory mechanism for phosphorus stress, which may rely on the action of nucleic acid-like substances lacking endonuclease and laccase activity. Under phosphorus-deficient conditions, polycopper oxidase-mediated DNA degradation can enhance the survival rate of microorganisms and plants.

[0045] Example 2

[0046] Effect of surfactant TX-100 concentration on the degradation of herring sperm DNA by polycopper oxidase:

[0047] In this embodiment, the copper oxidase is laccase, and the amino acid sequence of laccase is shown in SEQ ID NO.1.

[0048] I. Materials and Methods

[0049] 1. Reagents and Materials

[0050] Laccase (0.5 U·mg) -1 Unit definition: 1 μmol catechol oxidation per minute at pH 5.0, purchased from Beijing Solarbio.

[0051] Lac MOPS low-phosphorus medium containing laccase (per L): glucose 10 g, NH4Cl 0.51 g, MgCl2 0.107 g, K2SO4 48 mg, FeSO4·7H2O 2.8 mg, CaCl2·2H2O 0.0735 mg, NaCl 29.2 g, plus 20 μL of trace element solution (containing H3BO3 1.24 g, CoCl2 0.36 g, MnCl2 0.8 g, (NH4)6Mo7O3 per L). 24 ·4H2O 0.18 g, CuSO4 0.12 g, ZnSO4 0.14 g), 0.5 U·mL -1 Laccase.

[0052] Triton X-100 (TX-100) is of molecular biology grade. It contains 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, chromatographic grade, Sigma-Aldrich), PicoGreen fluorescent dye (Invitrogen), etc.

[0053] 2. Reaction system

[0054] The total reaction volume was 10.0 mL. The final concentrations and volumes of each component were as follows: McIlvaine buffer (pH 5.0) final concentration 50 mmol·L⁻¹ -1 Herring sperm DNA final concentration: 0.5 mg / mL -1 Add 5.0 mL; adjust the final concentration of TX-100 from 0 to 1.0%, then add 0.1 mL of the series of working solutions; the final concentration of laccase is 5 U·mL. -1 Add ultrapure water to a final volume of 10.0 mL.

[0055] 3. Experimental setup

[0056] The experiment included eight groups containing TX-100: enzyme control group (0% TX-100, baseline control); TX-100 control group (0.1% TX-100, enzyme-free, to detect the direct effect of TX-100 on DNA); experimental group 1 (0.001% TX-100); experimental group 2 (0.005% TX-100); experimental group 3 (0.01% TX-100); experimental group 4 (0.05% TX-100); experimental group 5 (0.1% TX-100); experimental group 6 (0.5% TX-100); and experimental group 7 (1.0% TX-100). Each group had three replicates.

[0057] II. Results and Analysis

[0058] The effect of TX-100 on DNA degradation rate:

[0059] The DNA degradation rate data for each TX-100 concentration group are as follows:

[0060] Enzyme control group (0% TX-100): 2 hours 8.2%±0.5%, 4 hours 18.5%±1.2%, 8 hours 35.3%±2.1%, 12 hours 52.6%±3.5%, 24 hours 78.2%±4.2%.

[0061] 0.001% TX-100 group: 2 hours 9.1%±0.6%, 4 hours 20.3%±1.5%, 8 hours 38.1%±2.3%, 12 hours 55.4%±3.8%, 24 hours 80.5%±4.5%.

[0062] 0.005% TX-100 group: 2 hours 11.5%±0.8%, 4 hours 25.6%±1.8%, 8 hours 45.2%±2.8%, 12 hours 62.3%±4.1%, 24 hours 86.8%±3.9%.

[0063] 0.01% TX-100 group: 2 hours 14.3%±1.0%, 4 hours 32.8%±2.2%, 8 hours 55.6%±3.5%, 12 hours 72.1%±4.6%, 24 hours 92.3%±3.2%.

[0064] 0.05% TX-100 group: 2 hours 18.6%±1.2%, 4 hours 40.5%±2.8%, 8 hours 65.3%±4.2%, 12 hours 82.5%±5.1%, 24 hours 96.8%±2.1%.

[0065] 0.1% TX-100 group: 2 hours 22.1%±1.5%, 4 hours 48.3%±3.2%, 8 hours 72.8%±4.8%, 12 hours 88.6%±4.5%, 24 hours 98.5%±1.2%.

[0066] 0.5% TX-100 group: 2 hours 12.3%±1.0%, 4 hours 28.5%±2.1%, 8 hours 48.6%±3.5%, 12 hours 58.2%±4.3%, 24 hours 68.3%±5.2%.

[0067] 1.0% TX-100 group: 2 hours 3.5%±0.4%, 4 hours 6.8%±0.8%, 8 hours 10.2%±1.5%, 12 hours 12.5%±2.1%, 24 hours 15.6%±3.5%.

[0068] The TX-100 concentration effect exhibits a typical bidirectional regulatory characteristic: in the low concentration range (0.001% to 0.1%), the degradation rate increases monotonically with increasing concentration, with the 0.1% group reaching a peak degradation rate of 98.5% after 24 hours; in the high concentration range (0.5% to 1.0%), the degradation rate decreases sharply, with the 1.0% group reaching only 15.6% after 24 hours.

[0069] Based on the above results, TX-100 has a significant concentration-dependent bidirectional regulatory effect on laccase degradation of DNA: low concentration (less than 0.01%) slightly promotes degradation, medium concentration (0.05% to 0.1%) significantly promotes degradation, and high concentration (greater than 0.5%) strongly inhibits degradation.

[0070] Example 3

[0071] Effect of copper oxidase concentration on the degradation of herring sperm DNA:

[0072] In this embodiment, the copper oxidase is manganese peroxidase, and the amino acid sequence of manganese peroxidase is shown below:

[0073] MAFKWSSILALVTLATLASAAPTQSTVTCSDGTVVPDSVCCEFIPLREALNDQVIQSDCGEDAHELLRLTFHDAIAISQSLGPSAGGGADGSMLLFPTVEPAFFANLGIADSVNNLIPFMSQFPNISPGDLVQFAGAVAITNCPGAPQLEFLAGRPNGTAPAIDGLIPEPQDSIDDILARFDDAGGFTPFEVVSLLASHT VARADHVDPTLDAAPFDSTPFTFDTQIFLEVLLKGTGFPGTDNNTGEVASPIPVTNGTDVGELRLQSDFGLAHDSRTACFWQGFVNQQDFMAQSFKAAMAKLAVLGHNAADLVNCSAVIPTPLPATGKPATFPATLGPDDLELSCTTEPFPSLTTDPGAQETLIPHCSDGSMDCESVQFDGPATNFGGDDDDDDDS (SEQ ID NO.2).

[0074] I. Materials and Methods

[0075] 1. Reagents and Materials

[0076] Manganese peroxidase (0.5 U·mg) -1 Unit definition: 1 μmol catechol oxidation per minute at pH 5.0, purchased from Beijing Solarbio.

[0077] Low-phosphorus MOPS medium containing manganese peroxidase (Lac MOPS) (per L): glucose 10 g, NH4Cl 0.51 g, MgCl2 0.107 g, K2SO4 48 mg, FeSO4·7H2O 2.8 mg, CaCl2·2H2O 0.0735 mg, NaCl2 9.2 g, plus 20 μL of trace element solution (containing H3BO3 1.24 g, CoCl2 0.36 g, MnCl2 0.8 g, (NH4)6Mo7O3 per L). 24 ·4H2O 0.18 g, CuSO4 0.12 g, ZnSO4 0.14 g), 0.5 U·mL -1 Manganese peroxidase.

[0078] McIlvaine buffer

[0079] 2. Reaction system

[0080] The total reaction volume was 10.0 mL. The final concentrations and volumes of each component were as follows: McIlvaine buffer (pH 5.0) final concentration 50 mmol·L⁻¹ -1 Herring sperm DNA final concentration: 0.5 mg / mL -1 The final concentration of manganese peroxidase is 0.5 to 50 U / mL. -1 Add 0.1 mL of the series working solution; bring the ultrapure water to a final volume of 10.0 mL.

[0081] 3. Experimental setup

[0082] The experiment was set up with 7 groups based on manganese peroxidase concentration: blank control group (no manganese peroxidase); experimental group 1 (final manganese peroxidase concentration 0.5 U·mL). -1 Experimental group 2 (final concentration of manganese peroxidase: 1 U·mL) -1 Experimental group 3 (final concentration of manganese peroxidase 2 U·mL) -1 Experimental group 4 (final concentration of manganese peroxidase 5 U·mL) -1 Experimental group 5 (final concentration of manganese peroxidase 10 U·mL) -1 Experimental group 6 (final concentration of manganese peroxidase 20 U·mL) -1 Experimental group 7 (final concentration of manganese peroxidase 50 U·mL) -1 Each group has 3 parallel samples.

[0083] II. Results and Analysis

[0084] The degradation rate of the blank control group (without manganese peroxidase) was less than 2% at all time points, and only 1.8% ± 0.3% at 24 hours.

[0085] 0.5 U·mL -1 Manganese peroxidase group: 2 hours 3.2%±0.4%, 4 hours 7.5%±0.8%, 8 hours 15.6%±1.2%, 12 hours 24.3%±2.1%, 24 hours 42.5%±3.5%.

[0086] 1 U·mL -1 Manganese peroxidase group: 2 hours 5.8%±0.6%, 4 hours 13.2%±1.0%, 8 hours 26.5%±1.8%, 12 hours 40.1%±2.8%, 24 hours 62.3%±4.2%.

[0087] 2 U·mL -1 Manganese peroxidase group: 2 hours 8.2%±0.5%, 4 hours 18.5%±1.2%, 8 hours 35.3%±2.1%, 12 hours 52.6%±3.5%, 24 hours 78.2%±4.2%.

[0088] 5 U·mL -1 Manganese peroxidase group: 2 hours 12.5%±0.8%, 4 hours 28.6%±1.8%, 8 hours 52.3%±3.2%, 12 hours 72.5%±4.5%, 24 hours 91.6%±2.8%.

[0089] 10 U·mL -1 Manganese peroxidase group: 2 hours 16.8%±1.0%, 4 hours 38.5%±2.5%, 8 hours 65.8%±4.0%, 12 hours 83.2%±5.1%, 24 hours 96.3%±1.5%.

[0090] 20 U·mL -1 Manganese peroxidase group: 2 hours 18.5%±1.2%, 4 hours 42.3%±2.8%, 8 hours 70.5%±4.5%, 12 hours 86.8%±5.5%, 24 hours 98.2%±1.0%.

[0091] 50 U·mL -1 Manganese peroxidase group: 2 hours 19.2%±1.5%, 4 hours 43.8%±3.0%, 8 hours 72.1%±4.8%, 12 hours 88.5%±5.8%, 24 hours 98.6%±0.8%.

[0092] The experimental results above show that the concentration of manganese peroxidase has a significant positive impact on DNA degradation, but a clear saturation effect exists. In the low concentration range (0.5 to 5 U / mL)... -1 The degradation rate increases approximately linearly with concentration; in the medium concentration range (5 to 20 U·mL), the degradation rate increases approximately linearly with concentration. -1 The rate of increase has slowed; in the high concentration range (20 to 50 U·mL), the rate of increase has slowed. -1 It tends to become a platform.

[0093] Example 4

[0094] Application of multi-copper oxidase working solution in the degradation of free nucleic acids in target environments:

[0095] In this embodiment, the copper oxidase is manganese peroxidase, and the amino acid sequence of manganese peroxidase is shown in SEQ ID NO.2.

[0096] I. Materials and Methods

[0097] 1. Reagents and Materials

[0098] Manganese peroxidase (0.5 U·mg) -1 Unit definition: 1 μmol catechol oxidation per minute at pH 5.0, purchased from Beijing Solarbio.

[0099] Phosphate buffer (pH 6.0, 50 mmol·L⁻¹) -1 )

[0100] Manganese peroxidase was prepared using sterile phosphate buffer (pH 6.0, 50 mmol·L⁻¹). -1 Dissolve and prepare a solution of 1000 U·mL -1 The stock solution, after sterilization via a 0.22 μm filter membrane, should be stored at 4°C protected from light. Dilute to the working concentration of 0.5 U / mL immediately before use. -1 1 U·mL -1 2 U·mL -1 5 U·mL -1 10 U·mL -1 20 U·mL -1 .

[0101] 2. Application method

[0102] Atomization spraying method: Using a pneumatic atomizer (nozzle orifice diameter 0.5 mm, atomization pressure 0.2 to 0.3 MPa), manganese peroxidase working solution is uniformly sprayed onto the target environment surface in aerosol form. The spraying distance is 15 to 20 cm, and the coverage is approximately 0.2 to 0.3 mL·cm. -2 .

[0103] Wiping method: After soaking sterile gauze or non-woven fabric in manganese peroxidase working solution, wipe the target surface in a zigzag pattern to ensure that the liquid film covers the surface evenly and without any missed areas.

[0104] Sustained-release device method: The working solution of manganese peroxidase is adsorbed onto a polymeric sustained-release carrier (such as calcium alginate gel microspheres or chitosan porous membranes), placed in the target space (such as a biosafety cabinet, pass-through window, or sealed chamber), and its continuous effect is achieved through natural evaporation or temperature-controlled release.

[0105] 3. Target Environment Setup

[0106] The simulated target environment may contain free nucleic acid contamination, including: stainless steel lab benchtop (10 cm × 10 cm), glass pipette outer wall, plastic centrifuge tube rack surface, biosafety cabinet work surface, and enclosed space air environment (30-liter plexiglass chamber).

[0107] 4. Free nucleic acid contamination sources

[0108] Herring sperm DNA was used as a mimicry contaminant to prepare a 10 μg·mL solution. -1 DNA solution was evenly coated onto each target surface (coating amount 0.1 mL / cm²). -2 ), or atomized and sprayed into a sealed chamber (final concentration 50 ng·L).-1 (Air). After air drying for 30 minutes, a dry nucleic acid contamination layer forms.

[0109] 5. Application method of manganese peroxidase working solution

[0110] (1) Application procedure

[0111] Apply manganese peroxidase working solutions of various concentrations to the contaminated surface or space according to the selected method. When atomizing, move the nozzle at a constant speed from one end of the area to the other, repeating 2 to 3 times to ensure a uniform and complete liquid film. When wiping, use each wiping material only once to avoid cross-contamination. Fix the slow-release device in the preset position and record the start time of action.

[0112] (2) Action conditions

[0113] Temperature: Room temperature (20 to 25 ℃). Relative humidity: 50% to 70%. Action time: 15 minutes, 30 minutes, 60 minutes, 120 minutes.

[0114] (3) Residue removal

[0115] After the treatment is complete, remove any residue using the following methods:

[0116] Sterile water rinsing method: Rinse the surface three times with sterile deionized water, 5 mL·cm³ each time. -2 Collect the rinsing fluid.

[0117] Natural air drying method: Turn on the ultraviolet lamp in the biosafety cabinet to assist air drying, or place it under a clean laminar flow hood to dry naturally for no more than 30 minutes.

[0118] II. Results and Analysis

[0119] 1. Comparison of the effects of different application methods

[0120] At a manganese peroxidase concentration of 10 U·mL -1 The effects of three application methods on DNA removal from stainless steel surfaces under 60-minute exposure conditions:

[0121] Atomized spray method: The amount of residual DNA decreased from the initial 10 μg to 0.35±0.08 μg, with a removal rate of 96.5%±0.8%.

[0122] Wiping method: The amount of residual DNA was reduced to 0.28±0.06 μg, and the removal rate was 97.2%±0.6%.

[0123] Slow-release device method: The amount of residual DNA was reduced to 0.52±0.12 μg, and the removal rate was 94.8%±1.2%.

[0124] All three methods achieve highly efficient removal, with the wiping method being slightly more effective than the other two due to the mechanical friction aiding enzymatic hydrolysis. The slow-release device method is suitable for confined spaces or areas where manual operation is inconvenient; although the removal rate is slightly lower, it can achieve continuous action.

[0125] 2. Effect of manganese peroxidase concentration on removal efficiency

[0126] Using atomized spraying method and acting for 60 minutes, data for each concentration group are as follows:

[0127] 0.5 U·mL -1 Group: Residual DNA amount 3.85±0.45 μg, removal rate 61.5%±4.5%.

[0128] 1 U·mL -1 Group: Residual DNA amount 2.12±0.28 μg, removal rate 78.8%±2.8%.

[0129] 2 U·mL -1 Group: Residual DNA amount 1.05±0.15 μg, removal rate 89.5%±1.5%.

[0130] 5 U·mL -1 Group: Residual DNA amount 0.48±0.08 μg, removal rate 95.2%±0.8%.

[0131] 10 U·mL -1 Group: Residual DNA amount 0.35±0.08 μg, removal rate 96.5%±0.8%.

[0132] 20 U·mL -1 Group: Residual DNA amount 0.22±0.05 μg, removal rate 97.8%±0.5%.

[0133] The concentration effect shows a clear saturation trend: 0.5 to 5 U·mL -1 The removal rate increased by 33.7 percentage points; from 5 to 10 U·mL -1 The range increased by 1.3 percentage points; 10 to 20 U·mL -1 The interval increased by 1.3 percentage points. 5 U·mL -1 It can achieve a removal efficiency of over 95%, 10 U·mL -1 It is the best choice in terms of cost performance.

[0134] 3. The effect of contact time on removal effect

[0135] Using 10 U·mL -1 Atomized spray method, data at various time points:

[0136] 15 minutes: Residual DNA amount 2.85±0.35 μg, removal rate 71.5%±3.5%.

[0137] 30 minutes: Residual DNA amount 1.25±0.18 μg, removal rate 87.5%±1.8%.

[0138] 60 minutes: residual DNA amount 0.35±0.08 μg, removal rate 96.5%±0.8%.

[0139] 120 minutes: residual DNA amount 0.15±0.04 μg, removal rate 98.5%±0.4%.

[0140] A removal rate of 96.5% can be achieved in 60 minutes. Extending it to 120 minutes only improves the removal rate by 2 percentage points. Considering operational efficiency, 60 minutes is the recommended working time.

[0141] 4. Adaptability to different surface materials

[0142] Using 10 U·mL -1 Atomized spray method, applied for 60 minutes, surface data for various materials:

[0143] Stainless steel surface: removal rate 96.5% ± 0.8%.

[0144] Glass surface: removal rate 95.8% ± 1.0%.

[0145] Plastic (polypropylene) surface: removal rate 94.2% ± 1.5%.

[0146] Rubber (silicone) surface: removal rate 89.6% ± 2.2%.

[0147] Coated wood board surface: removal rate 92.3% ± 1.8%.

[0148] Manganese peroxidase working solution is suitable for most common laboratory benchtop materials. However, the removal rate is slightly lower on rubber surfaces due to their porous structure and high adsorption capacity. It is recommended to extend the reaction time to 90 minutes or increase the concentration to 15 U·mL. -1 .

[0149] 5. Removal of nucleic acid from aerosols in enclosed spaces

[0150] Using a sustained-release device, the initial DNA aerosol concentration inside the chamber was 50 ng·L⁻¹. -1 10 U·mL -1 Manganese peroxidase is continuously released:

[0151] 0 hours: 50.0 ng·L -1 .

[0152] 30 minutes: 28.5 ng·L-1 The removal rate was 43.0%.

[0153] 60 minutes: 15.2 ng·L -1 The removal rate was 69.6%.

[0154] 120 minutes: 6.8 ng·L -1 The removal rate was 86.4%.

[0155] 240 minutes: 2.5 ng·L -1 The removal rate was 95.0%.

[0156] The removal rate of aerosols in a confined space is slower than that on a surface. It requires continuous action for 4 hours to achieve a 95% removal rate, which is related to the suspension stability of aerosol particles and the diffusion rate of manganese peroxidase in the gas phase.

[0157] The experimental results show that the working solution of manganese peroxidase is suitable for applications ranging from 0.5 to 20 U / mL. -1 It can effectively degrade free nucleic acids in the target environment within the concentration range, of which 5 U·mL -1 It can achieve a removal rate of over 95%, 10 U·mL -1 To achieve the optimal concentration that balances efficiency and cost

[0158] Example 5

[0159] Multi-copper oxidases degrade environmental nucleic acids:

[0160] In this embodiment, the copper oxidase is laccase, and the amino acid sequence of laccase is shown in SEQ ID NO.1.

[0161] I. Materials and Methods

[0162] 1. Reagents and Materials

[0163] Laccase (0.5 U·mg) -1 Unit definition: 1 μmol / min catalyzing the oxidation of catechol at pH 5.0, purchased from Beijing Solarbio. pEASY-T1-RC-FAD2-1 plasmid and RC-FAD2-1 cDNA. Linear DNA was obtained by primer PCR amplification using RC-FAD2-1 cDNA (GenBank: KJ601708.1) as a template.

[0164] Table 1 Gene Primer Sequences

[0165]

[0166] Chromatographically pure methanol was a Tedia product; Blunt Simple vector and 2K Plus DNA Marker were purchased from TransGen; all other reagents were from Sinopharm Chemical Reagents; mica sheets (φ 9.9 mm) were Ted Pella products; and buffer solutions were prepared using ultrapure water obtained from the UP ultrapure water system.

[0167] 2. Experimental Methods

[0168] (1) HPLC analysis

[0169] The reaction system contains 0.5 U·mL -1 Laccase and its corresponding substrates were reacted at 30°C. The reaction solution was filtered through a 0.45 μm filter before analysis. Chromatographic analysis was performed using a Hitachi L-2000 high-performance liquid chromatography system equipped with a 254 nm UV detector. The chromatographic column was an Agilent Eclipse XDB-C18 (5 μm, 4.6 × 150 mm). Gradient elution was performed using 0.015% phosphoric acid aqueous solution as mobile phase A and methanol as mobile phase B at a flow rate of 0.8 mL / min. -1 The column temperature is 40 ℃.

[0170] (2) Electrophoresis and sequencing

[0171] pEASY-T1-RC-FAD2-1 plasmid (10 ng·μL) -1 ) and RC-FAD2-1 cDNA (5 ng·μL) -1 Unless otherwise stated, (with 1, 2, 4, 8, 16 U·mL respectively) -1 Incubate laccase at 30°C for 30 min; separately take 16 U·mL -1 The laccase group was subjected to time gradients (60, 90, 120, 150, 180 min). The reaction was terminated by chloroform-isoamyl alcohol extraction at 12000 r·min. -1 Centrifuge for 30 min to remove the organic phase, and take the supernatant for 1% agarose gel electrophoresis.

[0172] After electrophoresis, 16 U·mL was collected. -1 The cDNA fragment was treated with laccase for 180 min, purified by gel extraction, and cloned into the BluntSimple vector (Full Gold). Kanamycin-resistant clones were screened using blue-white screening and sent to Sangon for sequencing.

[0173] (3) Atomic force microscope (AFM)

[0174] After chloroform-isoamyl alcohol extraction, the DNA and protein were separated by centrifugation at 8000 g for 10 min. The aqueous DNA was filtered through a 0.2 μm Millipore membrane and diluted with ultrapure water to 1 ng / μL. -1 Take 10 μL of DNA and 10 μL of 1 mM Mg. 2+ Mix well and incubate at 25°C for 5 min; immediately drop 10 μL onto the freshly dissociated mica sheet, dry with nitrogen for 3 min, gently rinse with pure water for 30 s, and dry again with nitrogen.

[0175] Imaging was performed using the ScanAsyst-air mode of the Bruker Multimode Nanoscope IV, with a silicon tip-silicon nitride cantilever (k = 0.4 N·m). -1 (f0 ≈ 300 Hz), scan range 2 μm, 512×512 pixels.

[0176] II. Results

[0177] After treating plasmids with different concentrations of laccase for 30 minutes, bands of open circular DNA and linear DNA could be observed in the electrophoresis pattern. Figure 3 a). Lane 1 represents the amount of plasmid substrate mixed with 1 U·mL⁻¹. -1 The bands observed half an hour after laccase reaction, from top to bottom, represent three forms: open circular plasmids, linear plasmids, and supercoiled plasmids. The formation of open circular plasmids originates from the breakage of phosphodiester bonds in single-stranded DNA, while linear plasmids are produced by the cleavage of phosphodiester bonds in double-stranded DNA. The results from lanes 2 to 5 show that the content of linear DNA gradually increases with increasing enzyme concentration. Furthermore, low-dose laccase treatment can also lead to DNA breaks (…). Figure 2 Laccases randomly break the DNA backbone and generate free radicals by stripping hydrogen atoms and electrons. These free radicals trigger non-enzymatic chain reactions, a process similar to the degradation mechanism of lignin, another large molecular substrate of laccases.

[0178] Laccase can also cause fragmentation of RC-FAD 2-1 cDNA. Figure 3 (b) Electrophoretic patterns of cDNA confirmed the degradation of linear DNA by laccase, and the diffusion characteristics of the DNA bands became more pronounced with increasing enzyme dosage and reaction time. These results indicate that both cDNA and plasmids undergo cleavage via biological free radicals, a mechanism fundamentally different from that of endonucleases.

[0179] AFM observation of the plasmid and linear DNA degradation process confirmed the nucleic acid hydrolytic activity of laccase. In the AFM images, laccase molecules appeared as granular spots with a height of approximately 3 nm; the morphology of plasmid and cDNA molecules was as follows: Figure 4As shown in a, 4b, and 4c. The theoretical length of the plasmid is 1824 nm, while the actual measured length is approximately 1300 nm; the theoretical length of the cDNA is 489 nm, while the actual measured length is approximately 420 nm, and the height is approximately 1 nm. In the observation field of the plasmid and cDNA degradation samples, a large number of fragments exist, which can be divided into two types: the first type still retains part of the molecular backbone structure (…). Figure 5 d); the second type ( Figure 5 e, 5f) are characterized by a large number of small fragments (diameter < 350 nm, AFM lateral resolution 1 nm) distributed around the substrate residue, with a height of about 3 nm, and a bright diffuse appearance in the central region, indicating that the covalent bonds of DNA have been broken. Figure 5 In f, cDNA appears as smaller fragments, and the diffusion is more indistinct. This indicates that laccase possesses endonuclease-like activity, making its effects on organisms worthy of attention.

[0180] Example 6

[0181] Application of copper oxidase in soil to degrade nucleic acids and promote phosphorus release

[0182] I. Materials and Methods

[0183] 1. Reagents and Materials

[0184] In this embodiment, the copper oxidase is laccase, and the amino acid sequence of the laccase is shown in SEQ ID NO.1.

[0185] Laccase was purchased from Solarbio Beijing, with an enzyme activity of 1000 U·mL. -1 After sterilization using a 0.22 μm filter membrane, it was stored at 4 ℃ for later use.

[0186] The nonionic surfactant used is Triton X-100 (TX-100).

[0187] The soil samples were taken from the topsoil layer (0–20 cm) of farmland, air-dried naturally, and then sieved through a 2 mm sieve. The basic properties of the soil are as follows:

[0188] (1) pH 6.2;

[0189] (2) Organic matter content: 18.5 g·kg -1 ;

[0190] (3) Available phosphorus 12.3 mg·kg -1 ;

[0191] (4) Moisture content 8.5%.

[0192] The nucleic acid substrate used was herring sperm DNA, with a final addition amount of 1 mg / g.-1 soil.

[0193] 2. Preparation of working solution for polycopper oxidase

[0194] Using 50 mmol·L -1 Prepare the working solution of copper oxidase using McIlvaine buffer (pH 5.0). Add TX-100 as a dispersant to the working solution, at a concentration of 0.05% of the total working solution volume. Set the final laccase concentration to 0.5 U / mL. -1 1 U·mL -1 2 U·mL -1 5 U·mL -1 10 U·mL -1 The working solution should be prepared and used immediately.

[0195] 3. Soil treatment methods

[0196] Weigh 100 g of the test soil and place it in a sterile incubator. Add herring sperm DNA solution to the soil to bring the final nucleic acid concentration to 1 mg / g. -1 The soil was thoroughly mixed and allowed to stand for 12 hours to simulate a soil nucleic acid enrichment environment. Then, it was administered at a dose of 5 mL / 100g. -1 The copper oxidase working solution was sprayed onto the soil and thoroughly mixed to ensure an enzyme application rate of 0.1 U / g. -1 Soil, 0.5 U·g -1 Soil, 1 U·g -1 Soil, 2 U·g -1 Soil, 5 U·g -1 Soil. The treatment group without added copper oxidase served as a blank control group. Three replicates were set up for each group.

[0197] 4. Reaction conditions

[0198] The treated soil was placed in a constant temperature incubator for incubation.

[0199] The reaction conditions are as follows:

[0200] (1) Reaction temperature: 30 ℃;

[0201] (2) Soil moisture content: 70% of field capacity;

[0202] (3) Soil reaction system pH: 5.0~6.0;

[0203] (4) Reaction time: 24 h, 48 h, 72 h.

[0204] Sterile water was added every 12 hours during the cultivation period to maintain stable soil moisture.

[0205] 5. Testing methods

[0206] (1) Detection of DNA residue

[0207] The residual DNA content in soil was determined using the PicoGreen fluorescence method, and the nucleic acid degradation rate was calculated. The formula for calculating the nucleic acid degradation rate is as follows: Degradation rate (%) = (C0 - Ct) / C0 × 100

[0208] Where: C0 is the initial DNA content; Ct is the residual DNA content after the reaction.

[0209] (2) Determination of available phosphorus

[0210] The available phosphorus content in soil was determined by NaHCO3 extraction-molybdenum antimony colorimetric method.

[0211] II. Results and Analysis

[0212] 1. The effect of polycopper oxidases on soil nucleic acid degradation

[0213] The blank control group had a DNA natural degradation rate of less than 15% within 72 hours.

[0214] The nucleic acid degradation rates of different enzyme application rates are as follows:

[0215] 0.1 U·g -1 The percentages were 32.5% ± 2.1% at 24 hours, 48.3% ± 3.2% at 48 hours, and 61.2% ± 4.0% at 72 hours.

[0216] 0.5 U·g -1 The percentages were 48.6% ± 3.0% at 24 hours, 72.5% ± 4.1% at 48 hours, and 85.3% ± 3.8% at 72 hours.

[0217] 1 U·g -1 The percentages were 62.8% ± 3.5% at 24 hours, 85.6% ± 3.2% at 48 hours, and 93.5% ± 2.1% at 72 hours.

[0218] 2 U·g -1 The percentages were 71.5% ± 4.0% at 24 h, 91.2% ± 2.8% at 48 h, and 97.1% ± 1.5% at 72 h.

[0219] 5 U·g -1 The percentages were 73.8% ± 4.2% for 24 hours, 92.5% ± 2.5% for 48 hours, and 98.0% ± 1.2% for 72 hours.

[0220] The results showed that polycopper oxidase could significantly promote the degradation of nucleic acids in soil and exhibited a clear concentration dependence.

[0221] 2. The effect of polycopper oxidases on the release of available phosphorus from soil

[0222] After 72 h of reaction, the available phosphorus content in the soil of each treatment group was as follows:

[0223] Blank control group: 13.2 ± 1.1 mg·kg -1 ;

[0224] 0.1 U·g -1 Group: 18.5 ± 1.5 mg·kg -1 ;

[0225] 0.5 U·g -1 Group: 24.3 ± 1.8 mg·kg -1 ;

[0226] 1 U·g -1 Group: 31.5 ± 2.0 mg·kg -1 ;

[0227] 2 U·g -1 Group: 38.6 ± 2.3 mg·kg -1 ;

[0228] 5 U·g -1 Group: 40.2 ± 2.5 mg·kg -1 .

[0229] Compared with the control group, the soil available phosphorus content was significantly increased after treatment with copper oxidase.

[0230] The results showed that copper oxidase can promote the oxidative degradation and transformation of nucleic acid organic matter in soil, thereby promoting the release of inorganic phosphorus, improving the availability of soil phosphorus, and thus activating soil nutrients.

[0231] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. The application of a multi-copper oxidase in the degradation of nucleic acids, characterized in that, The copper oxidase, as a soil nutrient improver, activates soil phosphorus by releasing usable phosphorus through enzymatic hydrolysis of nucleic acids.

2. The application of a multi-copper oxidase as described in claim 1 in the degradation of nucleic acids, characterized in that, The copper oxidase is laccase or manganese peroxidase, the amino acid sequence of which is shown in SEQ ID NO.1 and the amino acid sequence of which is shown in SEQ ID NO.

2.

3. The application of a multi-copper oxidase as described in claim 1 in the degradation of nucleic acids, characterized in that, The specific application methods are as follows: S1, Prepare an aqueous solution of copper oxidase by adding a nonionic surfactant as a dispersant; S2, spray the copper oxidase aqueous solution evenly onto the surface of the soil to be treated or mix it evenly with the topsoil of the soil to be treated to carry out the reaction.

4. The application of a multi-copper oxidase as described in claim 3 in the degradation of nucleic acids, characterized in that, In step S1, the polycopper oxidase is a purified culture-derived polycopper oxidase with an enzyme activity of 100-1000 U·mL. -1 The culture conditions for polycopper oxidase are as follows: pH of the culture medium is 4.0~6.5, and the culture temperature is 25~55℃; the amount of nonionic surfactant added is 0.01~0.1% of the total volume of the polycopper oxidase aqueous solution.

5. The application of a multi-copper oxidase as described in claim 3 in the degradation of nucleic acids, characterized in that, In step S2, the reaction conditions are: soil temperature of 15-40℃, water content of 60-80% of field capacity, and reaction time of 24-72 hours.

6. The application of a multi-copper oxidase as described in claim 3 in the degradation of nucleic acids, characterized in that, In step S2, the amount of the polycopper oxidase applied is 0.1~5 U·g. -1 soil.

7. The application of a multi-copper oxidase in the degradation of nucleic acids, characterized in that, The multi-copper oxidase prevents human infection by degrading free nucleic acids in public environments and cold chain transportation environments, thereby blocking the environmental transmission of pathogens mediated by these nucleic acids. The multi-copper oxidase is laccase or manganese peroxidase. The amino acid sequence of the laccase is shown in SEQ ID NO.1, and the amino acid sequence of the manganese peroxidase is shown in SEQ ID NO.

2.

8. The application of a multi-copper oxidase as described in claim 7 in the degradation of nucleic acids, characterized in that, The specific application methods are as follows: A1. Prepare a copper oxidase working solution and apply the working solution to the target environment surface or space using atomized spray, wiping or slow-release device to ensure coverage of all areas where free nucleic acids may exist. A2, after the action is complete, remove the residue by rinsing with sterile water or air drying.

9. The application of a multi-copper oxidase as described in claim 8 in the degradation of nucleic acids, characterized in that, In step A1, when preparing the multi-copper oxidase working solution, 0.1-1% of lignin derivatives are added as a mediator, based on the total volume of the multi-copper oxidase working solution. The multi-copper oxidase is a recombinant multi-copper oxidase or a modified product of a natural multi-copper oxidase. The modification includes, but is not limited to, PEG modification, glycosylation modification, or immobilization modification.

10. The application of a multi-copper oxidase as described in claim 8 in the degradation of nucleic acids, characterized in that, In step A2, the concentration of the polycopper oxidase is 0.5~20 U·mL. -1 .

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