Preparation method and trace element regulation method of protein degrading pentachloronitrobenzene pesticide residues
By extracting Bacillus polymyxa protein with 40% saturated ammonium sulfate and adding Fe2+, the problem of difficult degradation of pentachloronitrobenzene pesticide residues in ginseng was solved, achieving efficient and safe pesticide residue removal.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JILIN AGRICULTURAL UNIV
- Filing Date
- 2023-05-05
- Publication Date
- 2026-05-26
AI Technical Summary
Existing technologies are insufficient for efficiently degrading pentachloronitrobenzene pesticide residues in ginseng, and the role of trace elements in microbial degradation methods has not been reported.
Proteins of Bacillus polymyxa were extracted using 40% saturated ammonium sulfate, and the degradation of pentachloronitrobenzene was significantly improved by adding trace element Fe2+. The specific steps included strain preparation, protein extraction and trace element regulation.
It significantly improved the degradation ability of polymyxa Bacillus to pentachloronitrobenzene, achieving efficient, green and safe removal of pesticide residues.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to a method for preparing proteins that degrade pentachloronitrobenzene (PCNB) pesticide residues and a method for regulating trace elements. Specifically, it involves the use of 40% saturated ammonium sulfate to extract proteins from Bacillus polymyxa, which can be used to degrade pentachloronitrobenzene pesticide residues; and the use of trace element Fe. 2+ Significantly enhances the degradation of pentachloronitrobenzene by Bacillus polymyxa, and its application in the production and processing of high-quality ginseng products. Background Technology
[0002] Ginseng (Panax ginseng CAMeyer) is a perennial herb belonging to the genus Panax in the family Araliaceae. It can be used both as food and medicine, possessing various effects such as tonifying the middle energizer, replenishing qi and blood, calming the mind, and anti-tumor properties. With increasing health awareness, the demand for ginseng products is growing. Ginseng has a long growth cycle and is susceptible to ginseng diseases during cultivation. Pesticide control is a common method, but due to the slow degradation, long half-life, and bioaccumulation of organochlorine pesticides (PCNB) in plants, as well as improper use, Guo Luyao reported in 2021 that PCNB and other structurally stable organochlorine pesticides could still be detected in ginseng from different producing areas, with some even exceeding the limit standards. This seriously affects the quality and safety of ginseng products and restricts the development of the ginseng industry. Therefore, finding a method to quickly and efficiently degrade pesticide residues is urgent. Methods for degrading pesticide residues are mainly divided into physical, chemical, and biological methods. However, physical methods are inefficient, and chemical methods are prone to secondary pollution. Microbial degradation of pesticide residues, on the other hand, is cost-effective, efficient, and environmentally friendly, making it an important method for degrading pesticide residues in ginseng. The applicant previously demonstrated that the ginseng endophyte *Paenibacillus polymyxa* Pp-7250 has the effect of degrading PCNB pesticide residues, but the specific protein involved in its PCNB degradation is unclear, and there are no reports on how trace elements enhance the PCNB degradation effect of Pp-7250. Summary of the Invention
[0003] The purpose of this invention is to solve the problem of high pesticide residues in ginseng, and to provide a method for preparing protein that degrades pentachloronitrobenzene (PCNB) pesticide residues and a method for regulating trace elements. The strain used in this method is *Paenibacillus polymyxa*, which was deposited on February 5, 2013, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 7250. The method involves extracting *Paenibacillus polymyxa* protein with 40% saturated ammonium sulfate, which can be used to degrade pentachloronitrobenzene pesticide residues. The trace element Fe2+ significantly enhances the degradation effect of *Paenibacillus polymyxa* on pentachloronitrobenzene.
[0004] A method for preparing a protein that degrades pentachloronitrobenzene pesticide residues, and the strain used in the preparation method is *Paenibacillus polymyxa*, which was deposited on February 5, 2013, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 7250. The method involves extracting the *Paenibacillus polymyxa* protein with 40% saturated ammonium sulfate, which can be used to degrade pentachloronitrobenzene pesticide residues. The trace element Fe2+ significantly enhances the degradation of pentachloronitrobenzene by *Paenibacillus polymyxa*. The method specifically includes the following steps:
[0005] 1) Preparation of pesticide residue protein degraded by Bacillus polymyxa:
[0006] OD is extracted under aseptic conditions 600nm 10 mL of 0.8% Bacillus polymyxa bacterial suspension was transferred to a centrifuge tube and centrifuged at 4°C and 6000 rpm for 10 min. The supernatant was filtered through a 0.22 μm microporous membrane and transferred to a sterile 50 mL Erlenmeyer flask equipped with a magnetic stirrer. Sterile ammonium sulfate solid powder was then slowly added to the Erlenmeyer flask until the ammonium sulfate saturation in the supernatant reached 40%. The mixture was stirred with a magnetic stirrer until the ammonium sulfate solid powder was completely dissolved. After standing overnight in a refrigerator at 4°C, the precipitate was collected by centrifugation at 4°C and 12000 rpm for 30 min to obtain 40% saturated ammonium sulfate precipitated protein.
[0007] 2) Trace elements improve the pesticide degradation efficiency of Bacillus polymyxa:
[0008] Preparation of trace element Fe 2+ A culture medium with a concentration of 50 mg / LPDB is used to culture Bacillus polymyxa for 24 hours, which is a highly efficient pentachloronitrobenzene degrading bacterial solution.
[0009] The pesticide residue-degrading protein of the Bacillus polymyxa is a Bacillus polymyxa protein extracted with 40% saturated ammonium sulfate.
[0010] The culture medium for improving the pesticide degradation efficiency of Bacillus polymyxa is enriched with trace element Fe. 2+ PDB medium with a concentration of 50 mg / L.
[0011] The beneficial effects of this invention are:
[0012] The *Bacillus polymyxa* protein prepared in this invention significantly degrades pentachloronitrobenzene pesticide residues in ginseng. The *Bacillus polymyxa* protein extracted with 40% saturated ammonium sulfate exhibits pentachloronitrobenzene degradation activity. The trace element Fe... 2+It significantly enhances the degradation of pentachloronitrobenzene by Bacillus polymyxa. Attached Figure Description
[0013] Figure 1 Chromatograms showing the degradation rate of PCNB by Bacillus polymyxa proteins extracted with ammonium sulfate at different saturations; a: PBS without PCNB, b: PBS with PCNB only, c, d, e, f, and g are proteins extracted with PCNB at different saturated ammonium sulfate concentrations of 40%, 50%, 60%, 70%, and 80%, respectively.
[0014] Figure 2 The degradation rate of PCNB by Bacillus polymyxa proteins extracted with ammonium sulfate at different saturations was determined.
[0015] Figure 3 Chromatograms showing the effects of different trace elements on the degradation of PCNB by Bacillus polymyxa; a is the culture medium without PCNB, b is the culture medium with PCNB only, c is the control group of Bacillus polymyxa without trace elements, and d, e, f, and g are Bacillus polymyxa with added Cu2+, Fe2+, Zn2+, and Mn2+ respectively, plus PCNB.
[0016] Figure 4 The figure shows the effect of different trace elements on the degradation of PCNB by Bacillus polymyxa. Detailed Implementation
[0017] A method for preparing a protein that degrades pentachloronitrobenzene pesticide residues and a method for regulating trace elements are disclosed. The strain used in the preparation method is *Paenibacillus polymyxa*, which was deposited on February 5, 2013, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 7250. The method involves extracting the *Paenibacillus polymyxa* protein with 40% saturated ammonium sulfate, which can be used to degrade pentachloronitrobenzene pesticide residues. The trace element Fe2+ significantly enhances the degradation of pentachloronitrobenzene by *Paenibacillus polymyxa*. The specific steps include:
[0018] 1) Preparation of pesticide residue protein degraded by Bacillus polymyxa:
[0019] OD is extracted under aseptic conditions 600nm10 mL of 0.8% Bacillus polymyxa bacterial suspension was transferred to a centrifuge tube and centrifuged at 4°C and 6000 rpm for 10 min. The supernatant was filtered through a 0.22 μm microporous membrane and transferred to a sterile 50 mL Erlenmeyer flask equipped with a magnetic stirrer. Sterile ammonium sulfate solid powder was then slowly added to the Erlenmeyer flask until the ammonium sulfate saturation in the supernatant reached 40%. The mixture was stirred with a magnetic stirrer until the ammonium sulfate solid powder was completely dissolved. After standing overnight in a refrigerator at 4°C, the precipitate was collected by centrifugation at 4°C and 12000 rpm for 30 min to obtain 40% saturated ammonium sulfate precipitated protein.
[0020] 2) Trace elements improve the pesticide degradation efficiency of Bacillus polymyxa:
[0021] Preparation of trace element Fe 2+ A culture medium with a concentration of 50 mg / LPDB is used to culture Bacillus polymyxa for 24 hours, which is a highly efficient pentachloronitrobenzene degrading bacterial solution.
[0022] The pesticide residue-degrading protein of the Bacillus polymyxa is a Bacillus polymyxa protein extracted with 40% saturated ammonium sulfate.
[0023] The culture medium for improving the pesticide degradation efficiency of Bacillus polymyxa is enriched with trace element Fe. 2+ PDB medium with a concentration of 50 mg / L.
[0024] Experimental Example: A method for preparing a protein that degrades pentachloronitrobenzene pesticide residues:
[0025] 1. Materials and Instruments:
[0026] 1.1 Materials
[0027] Pentachloronitrobenzene was purchased from Dima Technology, batch number 15001; *Bacillus polymyxa* was obtained from our laboratory and deposited on February 5, 2013, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, accession number CGMCC No. 7250. Acetone (analytical grade, Beijing Chemical Plant), petroleum ether (analytical grade, Beijing Chemical Plant), n-hexane (chromatographic grade, Fisher), glucose, and agar (Sinopharm Group).
[0028] 1.2 Instruments and Equipment
[0029] Gas chromatograph (SHIMADZU GC-14C), electronic analytical balance (SHIMDZU AUY220), electronic constant temperature water bath (Yuyao Shangtong Temperature Control Instrument Factory HHs-6s).
[0030] 2. Experimental Methods:
[0031] 2.1 Preparation of pesticide residue protein degraded by Bacillus polymyxa
[0032] OD is extracted under aseptic conditions 600nm 10 mL of 0.8% *Bacillus polymyxa* bacterial suspension was transferred to a centrifuge tube and centrifuged at 4°C and 6000 rpm for 10 min. The supernatant was filtered through a 0.22 μm microporous membrane and transferred to a sterile 50 mL Erlenmeyer flask equipped with a magnetic stirrer. Sterile ammonium sulfate powder was then slowly added to the flask until the ammonium sulfate saturation in the supernatant reached 40%. The mixture was stirred with a magnetic stirrer until the ammonium sulfate powder was completely dissolved. After standing overnight at 4°C, the mixture was transferred to a centrifuge tube and centrifuged at 4°C and 12000 rpm for 30 min to collect the precipitate, yielding 40% saturated ammonium sulfate precipitated protein. Following the same method, 50%, 60%, 70%, and 80% saturated ammonium sulfate precipitated proteins were obtained sequentially. The precipitates were then analyzed using a 10 mM pH... Resuspend and dissolve the protein in 5 ml of pH 7.4 PBS buffer, transfer it to a sterile dialysis bag, and perform desalting dialysis until no white turbidity appears when BaCl2 solution is added, indicating complete dialysis. Transfer the completely dialyzed protein solution to sterile Erlenmeyer flasks, adjust the concentration of each dialysis protein to the same level (9.8 ml) with 10 mM pH 7.4 PBS buffer, and then add 0.2 mL of 10 μg / mL pentachloronitrobenzene solution to each flask. Simultaneously, set up controls with only pentachloronitrobenzene pesticide and controls with only dialysis protein. Each group was repeated three times. After thorough mixing, the mixture was incubated at 28℃ and 120 rpm for 24 h. Samples were then taken, and the pesticide residue content was determined by GC. The pesticide residue degradation rate was calculated, and pesticide residue-reducing proteins were screened.
[0033] Trace element screening to improve the pesticide degradation efficiency of Bacillus polymyxa:
[0034] Prepare separate formulations with added trace element Cu 2+ Fe 2+ Zn 2+ Mn 2+ Different culture media, with each trace element concentration of 50 mg / L, were used. 9.8 mL of *Bacillus polymyxa* culture, cultured for 24 h in each medium, was transferred to sterile Erlenmeyer flasks. 0.2 mL of pentachloronitrobenzene solution (10 μg / mL) was added to each flask, and the mixtures were thoroughly mixed. The flasks were then incubated at 28℃ and 120 rpm for 4 days before sampling. Pesticide residues were determined by GC. A control group without added trace elements was also included.
[0035] 3. Results and Analysis:
[0036] 3.1 Isolation and Activity Determination of Pesticide Residue Proteins Degraded by Bacillus polymyxa
[0037] from Figure 1 and Figure 2It can be seen that the effects of ammonium sulfate with different saturations on the degradation of pesticide residues by Bacillus polymyxa proteins vary. The activity of 40% saturated ammonium sulfate extract on the degradation of pesticide residues is significantly higher than that of 50% and 60% saturated ammonium sulfate extract (P<0.05), and the activity of 50% and 60% saturated ammonium sulfate extract on the degradation of pesticide residues is significantly higher than that of 70% and 80% saturated ammonium sulfate extract (P<0.05).
[0038] 3.2 Effects of different trace elements on the degradation of pesticide residues by Bacillus polymyxa
[0039] Depend on Figure 3 and Figure 4 It can be seen that different trace element treatments have different effects on the degradation of pesticide residues by Bacillus polymyxa, with element Fe having the most significant impact. 2+ Significantly higher than element Zn 2+ (P<0.05) Degradation rate of PCNB by *Bacillus polymyxa*, element Zn 2+ The degradation rate of PCNB by *Bacillus polymyxa* treated with Mn2+ was significantly higher than that of PCNB treated with Cu (P<0.05), and the degradation rate of PCNB by Mn2+ was significantly higher than that of Cu. 2+ The degradation rate of PCNB by *Bacillus polymyxa* was measured (P<0.05).
[0040] 4. Conclusion:
[0041] 40% saturated ammonium sulfate extract of Bacillus polymyxa protein has the effect of degrading pentachloronitrobenzene, and the trace element Fe 2+ It significantly enhances the degradation of pentachloronitrobenzene by Bacillus polymyxa.
Claims
1. A method for preparing a protein that degrades pentachloronitrobenzene pesticide residues, characterized in that: The strain used in this method is *Bacillus polymyxa*, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 7250. The method involves extracting *Bacillus polymyxa* protein with 40% saturated ammonium sulfate, which can be used to degrade pentachloronitrobenzene pesticide residues. The specific steps in preparing the *Bacillus polymyxa* protein are as follows: OD is extracted under aseptic conditions 600nm 10 mL of 0.8% Bacillus polymyxa bacterial culture was transferred to a centrifuge tube and centrifuged at 4°C and 6000 rpm for 10 min. The supernatant was filtered through a 0.22 µm microporous membrane and transferred to a sterile 50 mL Erlenmeyer flask equipped with a magnetic stirrer. Sterile ammonium sulfate powder was then slowly added to the Erlenmeyer flask until the ammonium sulfate saturation in the supernatant reached 40%. The mixture was stirred with a magnetic stirrer until the ammonium sulfate powder was completely dissolved. After standing overnight at 4°C, the mixture was centrifuged at 4°C and 12000 rpm for 30 min to collect the precipitate, yielding 40% saturated ammonium sulfate precipitated protein.