Use of zanthoxylum bungeanum peroxidase in preparation of sewage treatment reagent

Abstract

The application provides a zanthoxylum armatum peroxidase and use of the zanthoxylum armatum peroxidase in preparation of sewage treatment reagents. The zanthoxylum armatum peroxidase can obtain a good phenol removal rate at a lower enzyme activity and a lower purity, the fresh zanthoxylum armatum is cheap, the zanthoxylum armatum peroxidase is simple to extract, and a complex basic equipment is not needed, which shows that the zanthoxylum armatum peroxidase has a good application potential in environmental protection.

Description

Technical Field

[0001] This invention relates to the use of peroxidase from Sichuan pepper in the preparation of wastewater treatment reagents. Background Technology

[0002] Phenolic compounds, including phenol, cresol, aminophenol, dinitro-o-cresol, tannic acid, and pentachlorophenol, are malodorous substances that can enter the human body through the digestive tract, respiratory tract, and skin. They bind to proteins in cell protoplasm, causing cells to lose vitality. Phenolic compounds are also toxic to the nervous, urinary, and digestive systems. Phenolic pollution mainly originates from waste gases and wastewater discharged during the production processes of coking, oil refining, coal gas manufacturing, phenol production, insulation materials, pharmaceuticals, and papermaking, and has a significant impact on plant growth and human health.

[0003] Bamboo-leaf Sichuan pepper (Zanthoxylum armatum DC.) is the fruit of a plant belonging to the genus Zanthoxylum L. in the family Rutaceae. It is a commonly used medicinal and edible product. When used medicinally, the dried, mature fruit is typically used and is currently included in local standards in Sichuan, Hunan, and Guangxi provinces. When used as a spice, the nearly mature fruit is commonly used; due to its greenish-blue color, it is also known as "green Sichuan pepper" in southwestern China. Its fragrant aroma and lingering numbing sensation have made it popular among consumers.

[0004] Bamboo leaf pepper is highly susceptible to browning during harvesting and processing, severely impacting its edible quality and commercial value. Numerous studies have shown that polyphenol oxidase and peroxidase in the plant are the main enzymes causing browning in its fruits. Current research primarily focuses on the relationship between enzymatic browning and polyphenol oxidase in bamboo leaf pepper; however, detailed reports on the properties of peroxidase in bamboo leaf pepper are lacking, and there are no reports of its use for removing phenolic substances from wastewater. Summary of the Invention

[0005] To address the above problems, this invention provides the use of peroxidase from Sichuan pepper in the preparation of wastewater treatment reagents.

[0006] Furthermore, the peroxidase of *Zanthoxylum bungeanum* contains four proteins with molecular weights of 46.32, 39.41, 35.03, and 9.06 kDa, respectively.

[0007] Furthermore, the peroxidase of *Zanthoxylum bungeanum* is obtained by extracting *Zanthoxylum bungeanum* with phosphate buffer solution, adding 15% ammonium sulfate to the extract until saturated, performing three-phase extraction on the supernatant, dialyzing the middle layer of the extract, and freeze-drying to obtain the solid.

[0008] Furthermore, the bamboo leaf pepper was extracted with 10 times the amount of 0.05 mol / L phosphate buffer solution.

[0009] Furthermore, the three-phase extraction involves adding 45% ammonium sulfate to the supernatant until saturated, followed by the addition of tert-butanol.

[0010] Centrifuge to obtain the extract.

[0011] Furthermore, the wastewater treatment reagent is used to remove phenolic substances from wastewater.

[0012] The present invention also provides a peroxidase of Sichuan pepper, which contains four proteins with molecular weights of 46.32, 39.41, 35.03 and 9.06 kDa, respectively.

[0013] Furthermore, it is obtained by extracting bamboo leaves and Sichuan pepper with phosphate buffer solution, adding 15% ammonium sulfate to the extract until saturated, performing three-phase extraction on the supernatant, dialyzing the middle layer of the extract, and freeze-drying to obtain the solid.

[0014] Furthermore, the bamboo leaf pepper was extracted with 10 times the amount of 0.05 mol / L phosphate buffer solution.

[0015] Furthermore, the three-phase extraction involves adding 45% ammonium sulfate to the supernatant until saturated, followed by the addition of tert-butanol.

[0016] Centrifuge to obtain the extract.

[0017] Finally, this invention provides a method for removing phenolic substances from wastewater, which involves adding the aforementioned *Zanthoxylum bungeanum* peroxidase to the wastewater containing phenolic substances at a final concentration of 200–300 U / mL.

[0018] Furthermore, the final concentration of the peroxidase in the bamboo leaf pepper is 280 U / mL.

[0019] This invention utilizes a three-phase extraction method to extract peroxidase from Sichuan pepper. The extracted Sichuan pepper peroxidase exhibits better temperature and pH stability compared to commercially available horseradish peroxidase, higher phenol removal efficiency, and better phenol removal rate within a shorter time and a wider pH and temperature range.

[0020] The peroxidase from Sichuan pepper of this invention can achieve better phenol removal rate even at lower enzyme activity and lower purity. Moreover, fresh Sichuan pepper is inexpensive, and the peroxidase from Sichuan pepper is easy to extract without the need for complex basic equipment. This indicates that the peroxidase from Sichuan pepper has good application potential in environmental protection.

[0021] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0022] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0023] Figure 1 Effects of ammonium sulfate dosage on peroxidase activity and protein content in crude enzyme extract (A: Curves showing changes in enzyme activity and protein content in the precipitate; B: Curves showing changes in enzyme activity and protein content in the supernatant; C: Actual effect of three-phase extraction)

[0024] Figure 2 Overall protein identification results (A), peptide length distribution (B), and protein coverage distribution (C)

[0025] Figure 3 Protein molecular weight distribution

[0026] Figure 4 The overlapping sequence of peroxidase peptides between bamboo leaf pepper and other plant peroxidases

[0027] Figure 5 Actual molecular weight determination results of peroxidase protein from Zanthoxylum bungeanum (A: SDS-PAGE results of ZADTP; B: SDS-PAGE results of P1 and P2; C: Standard curve of labeling; D: Native-PAGE results of ZADTP, P1, and P2; MW is the standard protein, ZADTP is the peroxidase extract of Zanthoxylum bungeanum, and P1 and P2 are products separated by SuperTandex-75 gel filtration chromatography)

[0028] Figure 6 Affinity study of peroxidase substrates in Zanthoxylum bungeanum (A and guaiacol; B affinity study)

[0029] Figure 7 Optimal temperature, pH, and stability studies of ZADTP and HRP (A: Optimal pH study, B: ZADTP pH stability, C: HRP pH stability, D: Optimal temperature study, E: ZADTP temperature stability, F: HRP temperature stability; ZADTP: Zanthoxylum bungeanum peroxidase; HRP: Horseradish peroxidase)

[0030] Figure 8 Model fitting results (A: one-step deactivation model; B: parallel deactivation model; C: tandem deactivation model; D: Arrhenius formula fitting results)

[0031] Figure 9 Effects of enzyme activity on phenol scavenging (A: Sichuan pepper peroxidase; B: horseradish peroxidase)

[0032] Figure 10 Effect of hydrogen peroxide concentration on phenol removal efficiency

[0033] Figure 11 The effect of pH value on phenol removal efficiency

[0034] Figure 12 The effect of temperature on phenol removal efficiency (A: phenol removal rate of bamboo leaf and Sichuan pepper peroxidase at different temperatures; B: phenol removal rate of horseradish peroxidase at different temperatures)

[0035] Figure 13 Effect of phenol concentration on clearance rate

[0036] Figure 14 The actual effect of peroxidase on phenol removal

[0037] Figure 15 Comparison of the detoxification effects of ZADTP and HRP peroxidase in Chinese prickly ash (root (C, D) and stem (A, B) length growth of plants after ZADTP and HRP treatment at 5 mmol / L and 10 mmol / L phenol concentrations, actual photos of seedling growth (E) (Note: different letters on the bars depict significant differences, p<0.05)) Detailed Implementation

[0038] Example 1: Study on the properties of peroxidase in bamboo leaf pepper and its ability to remove and detoxify phenol wastewater

[0039] 1. Experimental Materials

[0040] Main reagents: horseradish peroxidase, Coomassie brilliant blue, bovine serum albumin, dithiothreitol, acetonitrile, trifluoroacetic acid, Shanghai Maclean Biochemical Technology Co., Ltd.; phenol, 4-aminopyridoxine, sodium bicarbonate, tris(hydroxymethyl)aminomethane hydrochloride, glycine, hydrochloric acid, acetic acid, sodium acetate, sodium dihydrogen phosphate, disodium hydrogen phosphate, Chengdu Kelong Chemical Reagent Co., Ltd.; the remaining reagents are the same as those used in 2.1.1.

[0041] Main instruments: NanoElute ultra-high performance liquid chromatography, Capillary ion source, Brütek GmbH, Germany.

[0042] 2. Methods for enzyme activity determination and protein content determination

[0043] Peroxidase activity assay: Take 1 mL of 25 mmol / L guaiacol and H2O2 into test tubes, add 3 mL of buffer solution, and finally add 0.5 mL of crude enzyme extract to start the reaction. Quickly pour the mixture into a cuvette and measure the rate of increase of absorbance at 470 nm. Define one unit of enzyme activity as an increase of 0.01 absorbance per minute. Repeat the assay 3 times.

[0044] Protein content determination: The Bradford method was used to determine the protein content in the supernatant and precipitate. The specific steps were as follows: 100 mg of Coomassie Brilliant Blue was dissolved in 50 mL of 95% ethanol solution. After shaking well, 100 mL of 14.63 mol / L concentrated phosphoric acid solution was slowly added. After mixing well, the solution was diluted to 1000 mL with double-distilled water at pH 7.0 to obtain Bradford reagent. Seven test tubes were prepared. 0.00 mL, 0.01 mL, 0.02 mL, 0.04 mL, 0.06 mL, 0.08 mL, and 0.1 mL of standard bovine serum albumin solution (1 mg / mL) were added to each tube, and the solution in each tube was diluted to 0.1 mL with double-distilled water at pH 7.0. 5 mL of Bradford reagent was added. The mixture was thoroughly mixed, and the absorbance of each tube was measured at 595 nm. The absorbance was plotted on the ordinate, and the protein concentration on the abscissa. The standard curve is obtained as Y4 = 7.8778X4 + 0.0397.

[0045] R 2 =0.9999 (Y4: absorbance, X4: bovine serum albumin content mg / mL); total protein is calculated as bovine serum albumin.

[0046] 3. Extraction of peroxidase from Sichuan pepper (Zanthoxylum bungeanum)

[0047] Crude peroxide enzyme solution of Sichuan pepper: Take 100g of Sichuan pepper sample and add 1000mL of 0.05mol / L phosphate buffer solution (containing 2% polyvinylpyrrolidone, pH 6.5), homogenize, let stand for 10min, filter with four layers of gauze, centrifuge the filtrate at 10000r / min for 10min to obtain crude enzyme extract.

[0048] Investigation of ammonium sulfate dosage: Before three-phase extraction, the effects of 10%–60% ammonium sulfate dosage on protein content and activity in the crude enzyme were investigated using crude enzyme solution. Figure 1 As shown in Figure A, the protein content and enzyme activity in the precipitate gradually increased with increasing ammonium sulfate concentration. The highest enzyme activity and protein content were observed when the ammonium sulfate concentration was 0.6 g / mL. When the ammonium sulfate concentration exceeded 0.3 g / mL, the enzyme activity in the precipitate increased rapidly. Clearly, peroxidase can be extracted from the crude enzyme solution of *Zanthoxylum bungeanum* when the ammonium sulfate concentration is controlled between 0.3 g / mL and 0.6 g / mL. Figure 1 As shown in Figure B, the POD activity in the supernatant gradually decreased with increasing ammonium sulfate concentration. The enzyme activity decreased even more rapidly when the ammonium sulfate concentration exceeded 0.15 g / mL. Comprehensive analysis indicates that an ammonium sulfate concentration of 0.15 g / mL effectively removed most of the impurities from the crude enzyme solution, while also minimizing the impact on peroxidase activity.

[0049] Three-phase extraction: The crude enzyme solution was saturated with 15% ammonium sulfate (previously determined), and then centrifuged (4000 rpm, 10 min) to remove the lower precipitate. The supernatant was further processed by three-phase extraction: 45% ammonium sulfate was added again for saturation, followed by the addition of tert-butanol (1:1 v / v) to the ammonium sulfate-saturated supernatant, and then centrifuged again to promote phase separation. The intermediate precipitate was collected, dialyzed to remove salt, and lyophilized to obtain the preliminarily purified protein after three-phase extraction. The actual extraction effect was as follows: Figure 1 As shown in C. The ratio of total enzyme activity in the purified extract to total enzyme activity in the unpurified extract is the peroxidase recovery rate; specific activity is the total enzyme activity per milligram (mg) of protein; purification fold is the ratio of specific activity after purification to specific activity before purification; the determination results are shown in Table 1.

[0050] Table 1: Summary of peroxidase recovery and purification fold results in crude extract

[0051]

[0052] Table 1 shows that after extraction using the three-phase extraction method, the peroxidase in *Zanthoxylum bungeanum* was purified by a factor of 10.74, with a recovery rate of 99.03% and an enzyme activity ratio of 44990.23 U / mg. In summary, the three-phase extraction method combined with ammonium sulfate precipitation can extract peroxidase with a high purification factor from *Zanthoxylum bungeanum* without affecting the recovery rate.

[0053] 4. Investigation on the properties of peroxidase in Sichuan pepper (Zanthoxylum bungeanum)

[0054] 4.1 Molecular weight analysis of peroxidase in Zanthoxylum bungeanum

[0055] To understand the main protein composition of the three-phase extract of Sichuan pepper, this study used UPLC-MS / MS to further purify the proteins obtained after three-phase extraction. The specific steps are as follows:

[0056] (1) Protein extraction: The sample was taken out from -80℃, and an appropriate amount of sample was weighed and added to 4 times the volume of lysis buffer (1% SDS, 1% protease inhibitor). The sample was lysed by sonication. The sample was centrifuged at 12000g for 10 min at 4℃. The supernatant was transferred to a new centrifuge tube and the protein concentration was determined using a BCA kit.

[0057] (2) Trypsin digestion: Equal amounts of protein from each sample were digested, and the volumes were adjusted to be consistent using lysis buffer. One volume of pre-chilled acetone was added, vortexed, and then four volumes of pre-chilled acetone were added. The mixture was precipitated at -20°C for 2 hours. The precipitate was centrifuged at 4500g for 5 minutes, the supernatant was discarded, and the precipitate was washed twice with pre-chilled acetone. After drying the precipitate, TEAB was added to a final concentration of 200 mmol / L, and the precipitate was sonicated to disperse it. Trypsin was added at a ratio of 1:50 (protease: protein, m / m), and digestion was carried out overnight. Dithiothreitol (DTT) was added to a final concentration of 5 mmol / L, and the mixture was reduced at 56°C for 30 minutes. Iodoacetamide (IAA) was then added to a final concentration of 11 mmol / L, and the mixture was incubated at room temperature in the dark for 15 minutes.

[0058] (3) Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis: Peptides were dissolved in mobile phase A of the LC system and then separated using a NanoElute ultra-high performance liquid chromatography (UHPLC) system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was a solution containing 0.1% formic acid and 100% acetonitrile. The LC gradient settings were: 0-72 min, 7%–24% B; 72-84 min, 24%–32% B; 84-87 min, 32%–80% B; 87-90 min, 80% B, with the flow rate maintained at 400 mL / min. After separation by the UHPLC system, the peptides were injected into the Capillary ion source for ionization and then analyzed by a TimsTOF Pro mass spectrometer. The ion source voltage was set to 1.75 kV, and the precursor ion and secondary fragments of the peptides were detected and analyzed using high-resolution TOF. The secondary mass spectrometry scan range was set to 100–1700. The data acquisition mode used was Parallel Accumulation Serial Fragmentation (PASEF). After a primary mass spectrometer acquisition, 10 secondary spectra were acquired in PASEF mode for precursor ion charge numbers in the range of 0–5. The dynamic exclusion time for tandem mass spectrometry was set to 30 seconds to avoid repeated scanning of precursor ions.

[0059] (4) Data Processing: The obtained secondary mass spectrometry data were searched using Maxquant (v1.6.15.0). The search database consisted of protein sequences of Zanthoxylum nitidum plants and some plant peroxidase protein sequences obtained from Uniprot, totaling 1260 sequences. A common contamination library was added to the data to eliminate contaminating proteins in the identification results. The restriction enzyme digestion method was set to Trypsin / P; the number of missed cleavage sites was set to 2; the minimum peptide length was set to 7 amino acid residues; the maximum number of peptide modifications was set to 5; the mass error tolerance for primary precursor ions was set to 20 ppm; and the mass error tolerance for secondary fragment ions was also set to 20 ppm. Finally, the data obtained from the library search were further processed. The accuracy of identification at the spectrum, peptide, and protein levels was set to 1%, and the identification of a protein required the presence of at least one unique peptide.

[0060] The results of the identification are as follows Figure 2 As shown, a total of 158,294 spectra were generated during the mass spectrometry detection process. Among them, 172 were valid spectra (matched with theoretical secondary mass spectrometry), 143 were identified peptide sequences (resolved from the results), 27 were identified from the matching results, and 23 proteins were identified. Most peptides were distributed between 7 and 20 amino acids, which conforms to the general rules of enzymatic digestion and mass spectrometry fragmentation. The lengths of the identified peptides met the quality control requirements, and the coverage of most proteins by the identified peptides was below 30%.

[0061] The protein molecular weight distribution results are shown below. Figure 3 The results showed that after preliminary purification by three-phase extraction, the extract still contained other proteins. Their molecular weights were distributed across all phases, with most proteins ranging from 1 kDa to 90 kDa. This indicates that preliminary purification by three-phase extraction has a certain enrichment effect on proteins.

[0062] As shown in Table 2, the identified proteins partially overlap with the peptides of peroxidase proteins found in rapeseed, mustard, water celery, and lychee, with molecular weights of 34.207 kDa, 33.378 kDa, 37.658 kDa, and 8.6010 kDa, respectively. The overlapping peptide segments are shown in Table 2. Figure 4 .

[0063] Table 2. Protein identification results

[0064]

[0065] (5) To determine the composition of peroxidase in the three-phase extract, the extract was further separated using a SuperTandex-75 gel column. The separation results are shown in the figure. Two activity peaks, P1 and P2, were obtained. This result also shows that the peroxidase of Chinese prickly ash has isoenzymes with different molecular weights.

[0066] (6) Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the purity and molecular weight of the extracted peroxidase, while native polyacrylamide gel electrophoresis (native-PAGE) was used to determine the presence of peroxidase in the extract. For example... Figure 5 As shown in Figure A, the SDS-PAGE results of the three-phase extract of Zanthoxylum bungeanum (ZADTP) showed several bands, indicating that peroxidase is not the only protein in ZADTP. The product isolated by SuperTandex-75 showed three bands for activity peak 1 (P1) at 35-50 kDa, and one band for activity peak 2 (P2) at approximately 10 kDa. Figure 5 B). Meanwhile, P1 and P2 were also present in the ZADTP electrophoresis bands but not separated, indicating that SuperTandeX-75 can be used for the separation of peroxidase from *Zanthoxylum bungeanum*. Compared with standard proteins ( Figure 5 C) In comparison, the molecular weights were 46.32, 39.41, 35.03, and 9.06 kDa, similar to the results obtained by UPLC-MS / MS. However, due to the similar molecular weights of proteins in P1, and the lower protein content in the two lighter bands, the latter two bands could not be well separated. Figure 5 As shown in Figure D, after active staining, different bands appeared at different positions in the native-PAGE, which further illustrates the presence of peroxidase in Sichuan pepper.

[0067] 4.2 Investigation on the substrate specificity of peroxidase in Zanthoxylum bungeanum

[0068] The substrate specificity of the enzyme activity was investigated, and the specific steps are as follows. In the enzyme activity assay system, the concentration of guaiacol was fixed at 20 mmol / L, and the changes in enzyme activity were measured at hydrogen peroxide concentrations ranging from 1 mmol / L to 40 mmol / L. Similarly, the concentration of hydrogen peroxide was fixed at 20 mmol / L, and the changes in enzyme activity were measured at guaiacol concentrations of 20 mmol / L and peroxide concentrations ranging from 1 mmol / L to 40 mmol / L. The Michaelis constant (Km) and the maximum reaction rate (Vmax) of the *Zanthoxylum bungeanum* peroxidase under these conditions were calculated using Lineweaver-Burk with the derivative of the reaction rate as the ordinate and the substrate concentration as the abscissa. The results are shown below. Figure 6 .

[0069] from Figure 6It can be seen that within a certain concentration range, the reaction rate of peroxidase in *Zanthoxylum bungeanum* increases with increasing substrate concentration, and then tends to be constant. With a fixed guaiacol concentration of 20 mmol / L and hydrogen peroxide concentration in the range of 0–40 mmol / L, the enzyme-catalyzed reaction rate increases with increasing substrate concentration, and the reaction rate tends to be constant when the hydrogen peroxide concentration is greater than 15 mmol / L. With a fixed hydrogen peroxide concentration of 20 mmol / L, the reaction rate tends to be constant when the guaiacol concentration is greater than 30 mmol / L. The intermediate reaction complex theory can explain the relationship between substrate concentration and reaction rate. Under constant enzyme concentration, when the substrate concentration is low, the enzyme is not saturated with the substrate, and the enzyme-catalyzed reaction rate depends on the substrate concentration. As the substrate concentration increases, more enzyme-substrate intermediate complexes are formed, and the reaction rate is related to the concentration of the enzyme-substrate intermediate complex. When the substrate concentration continues to increase, the enzyme in the solution is completely saturated, and even if the substrate concentration is increased, no more enzyme-substrate intermediate complexes can be formed, and the reaction rate reaches its maximum value.

[0070] Peroxidase-catalyzed enzymatic reactions are two-substrate reactions. By fixing the concentration of one substrate, the effect of different concentrations of the other substrate on the reaction rate is measured. Using the Lineweaver-Burk double reciprocal plot method, two double reciprocal curves can be obtained, with the x-intercept at -1 / Km and the y-intercept at 1 / Vmax. From the double reciprocal curves of *Zanthoxylum bungeanum* peroxidase, it can be determined that when the H2O2 concentration is constant, the Km value for the enzyme to guaiacol is 35.67 mmol / L, and the Vmax value is 5000 U / mL. When the guaiacol concentration is constant, the Km value for H2O2 is 27.5 mmol / L, and the Vmax value is 4000 U / mL. The Km value is the substrate concentration at which the enzymatic reaction rate reaches half of the maximum reaction rate and can represent the affinity between the enzyme and the substrate. Therefore, it can be seen that H2O2 has a greater affinity for *Zanthoxylum bungeanum* peroxidase than guaiacol.

[0071] 4.3 Temperature and pH stability study of peroxidase in Zanthoxylum bungeanum

[0072] pH is a crucial factor determining enzyme activity because it can affect the ionization state of amino acids or substrates. Studies have reported that the optimal pH for an enzyme depends on the substrate used and the source of the enzyme. In this study, the effect of pH on the peroxidase activity of *Zanthoxylum bungeanum* was analyzed within the pH range of 2.0–9.0 using guaiacol and H2O2 as substrates. The specific steps are as follows: The enzyme solution obtained after preliminary purification via three-phase extraction was used, and the peroxidase activity was measured at 25℃ under different pH conditions (2.0–10.0). The highest enzyme activity was set as 100%, and the optimal pH was determined. The crude lyophilized enzyme powder was prepared with a buffer solution of pH 2.0–10.0, and enzyme activity was measured every 40 minutes for 4 hours. An untreated enzyme solution was used as a control, with relative enzyme activity at 100%. The results are as follows: Figure 7 .

[0073] Depend on Figure 7 A shows that peroxidase activity initially increases and then decreases with increasing pH. The highest peroxidase activity was observed in *Zanthoxylum bungeanum* at pH 6.0, while the peroxidase activity in *Hippophae rhamnoides*, a different plant source, was even higher at pH 7.0. This indicates that peroxidases from different plant sources have different properties. A comparison of the pH stability of peroxidases from *Zanthoxylum bungeanum* and *Hippophae rhamnoides* is shown in [the table below]. Figure 7 B, 5C. Figure 7 C indicates that horseradish peroxidase activity remained above 74% after 4 hours in a buffer solution with pH 5.0–8.0; the activity was above 23% at pH 4.0–10.0; and it was completely inactivated at pH 2.0–3.0. Figure 7 As shown in B, the peroxidase from *Zanthoxylum bungeanum* is stable within a pH range of 5.0–8.0. After 4 hours in a buffer solution with a pH of 5.0–8.0, its enzyme activity remained above 80%. After 4 hours of treatment within a pH range of 3.0–10.0, the peroxidase activity remained above 26%. However, at pH 2.0, the peroxidase was completely inactivated after 4 hours. In summary, the peroxidase from *Zanthoxylum bungeanum* exhibits a wider pH adaptability and better pH stability than commercial peroxidases.

[0074] The optimal temperature for enzyme activity often depends on the applied experimental conditions. Generally, the rate of enzyme-catalyzed reaction gradually decreases with increasing temperature, and under mild conditions, the native structure of the enzyme will change due to folding or unfolding. This study investigated the temperature stability of peroxidase from *Zanthoxylum bungeanum*, and the detailed procedures are as follows: The activity of peroxidase from *Zanthoxylum bungeanum* was measured at different temperatures (20℃~90℃), with the highest enzyme activity set at 100%. The relative activity of peroxidase at each temperature was calculated to determine the optimal temperature. The purified enzyme solution was placed under different temperature conditions (20℃~90℃), and samples were taken at 40 min, 80 min, 120 min, 160 min, 200 min, and 240 min to measure enzyme activity. An untreated enzyme solution was used as a control, with a relative enzyme activity of 100%.

[0075] like Figure 7 As shown in D. The optimal temperatures for peroxidase in *Zanthoxylum bungeanum* and *horseradish* are similar, both exhibiting the highest enzyme activity at 50℃. The results of the temperature stability study are as follows: Figure 7 E, F. The peroxidase from *Zanthoxylum bungeanum* remained stable within a temperature range of 30℃ to 60℃, while the peroxidase from *Hippophae rhamnoides* remained stable only within 30℃ to 50℃. *Zanthoxylum bungeanum* peroxidase retained 35% activity after treatment at 70℃ for 4 hours, while the activity of *Hippophae rhamnoides* peroxidase decreased to 25% after treatment at 60℃ for 4 hours and was completely inactivated after treatment at 70℃. This indicates that *Zanthoxylum bungeanum* peroxidase has better temperature stability compared to commercial peroxidases.

[0076] 4.4 Thermodynamics of Peroxidase in Bamboo Leaf Pepper

[0077] Heat treatment to inactivate enzymes is a common pretreatment method in food processing. This invention uses one-step inactivation, parallel inactivation, and tandem inactivation models to fit the heat inactivation data of *Zanthoxylum bungeanum* peroxidase at 65℃, 75℃, and 85℃, respectively. The specific expressions of the one-step inactivation, parallel inactivation, and tandem inactivation models are as follows: One-step inactivation model: When the enzyme exists only in a "natural state" and a "completely inactivated state" during heat inactivation, it can be represented by a one-step inactivation model. That is... This can be expressed by the following equation: The fitting results are shown below. Figure 8 A. Parallel inactivation model: When an enzyme consists of several enzymes with different thermostabilities, and each component conforms to a one-step inactivation model, i.e. This can be expressed by the following equation: The fitting results are shown below. Figure 8 B. Tandem inactivation model: When enzyme inactivation requires multiple transition states, a tandem inactivation model can be used to represent this process. This can be expressed by the following equation: The fitting results are shown below. Figure 8 C. In the above formula, k dk d1 and k d2 These are the deactivation rate constants of the reaction, A0 and A1, respectively. t These are the enzyme activities before and after heat treatment.

[0078] The specific parameter results for the fitting of the three models are shown in Table 3. At lower temperatures, the one-step inactivation model showed a poor fit, with a lower coefficient of determination (R²). 2 The R-value is only 0.79960, while the R-value of the inactivation model is much higher. 2 The values ​​were all above 0.98 at all three temperatures, indicating that the peroxidase inactivation was more consistent in the serial inactivation model. This may be due to the presence of several peroxidases with different molecular weights in *Zanthoxylum bungeanum*, and the different thermosensitive properties of these peroxidases. Therefore, the thermal inactivation of peroxidase in *Zanthoxylum bungeanum* progressed from the initial active state E0 to the intermediate state E... d1 to inactive state E d2 Two consecutive first-order reactions. In the serial inactivation model, k d1 At the same temperature, it is always less than k. d2 Therefore, the first inactivation process of peroxidase is a rate-limiting step, which determines the rate at which the enzyme is inactivated.

[0079] Table 3: Results of the one-step inactivation model and the tandem inactivation model

[0080]

[0081]

[0082] According to the Arrhenius equation, the activation energy (Ea) of an enzyme can be calculated from the slope of the Arrhenius diagram, using the following formula: (Ea is the activation energy, R is the gas constant (8.314 J·mol⁻¹)) -1 K -1 (T is temperature (Kelvin), and k is the inactivation rate constant). The effect of temperature on the enzyme inactivation rate is related to Ea. The larger the Ea, the higher the energy barrier that the enzyme needs to overcome for inactivation, and the better the enzyme's thermal stability.

[0083] The rate constant (k) obtained through fitting d1 and k d2 ) is used in the Arrhenius formula ( Figure 8 D). In this study, the activation energy required to inactivate the peroxidase in *Zanthoxylum bungeanum* was E. d1 =197.76kJ / mol and E d2=101.89 kJ / mol. Therefore, the complete inactivation of peroxidase in *Zanthoxylum bungeanum* requires 299.65 kJ / mol of energy, further demonstrating the temperature stability of *Zanthoxylum bungeanum* peroxidase. 4.5 Effects of Chemical Reagents on the Activity of Peroxidase in *Zanthoxylum bungeanum*

[0084] This study analyzed the effects of common enzyme inhibitors, surfactants, and metal ions at concentrations ranging from 0.1 mmol / L to 10 mmol / L on the peroxidase activity of *Zanthoxylum bungeanum*. The detailed procedure was as follows: different concentrations of inhibitors (citric acid, ascorbic acid, salicylic acid, and sodium sulfite) and metal ions (NaCl, K₂SO₄, CaCl₂, MgSO₄, ZnSO₄, CuSO₄, and MnSO₄) were added to the enzymatic reaction system to achieve concentrations of 0.1 mM, 1 mM, and 10 mM, respectively. The enzyme solution was mixed with the different concentrations of compounds and allowed to stand for 1 hour, after which the residual enzyme activity was measured. Enzyme activity measured in the absence of metal ions or inhibitors served as a blank control, and residual enzyme activity was defined as 100%. The results are shown in Table 4.

[0085] Table 4: Effects of metal ions and chemical reagents on peroxidase activity

[0086]

[0087]

[0088] Table 4 shows that most enzyme inhibitors and surfactants inhibited the peroxidase activity of *Zanthoxylum bungeanum*. Ascorbic acid, salicylic acid, and citric acid showed the best inhibitory effects on peroxidase activity, completely inhibiting it at concentrations above 1 mmol / L. Sulfite and sodium dodecyl sulfate (SDS) followed, with inhibition rates of 97.18% and 54.67% respectively at a concentration of 10 mmol / L. Table 4 also shows that the peroxidase activity of *Zanthoxylum bungeanum* is less affected by common metal ions, maintaining above 68% at different metal ion concentrations. This stability to metal ions is beneficial for its practical application.

[0089] 4.6 Summary

[0090] Unlike traditional protein purification and separation techniques, three-phase extraction selectively concentrates proteins into a single phase, achieving high recovery rates (99.03%) and purification folds (10.74). It is also lower in cost, involves fewer steps, and is less time-consuming, making it suitable for large-scale industrial extraction of crude peroxidase from *Zanthoxylum bungeanum*. Substrate specificity studies showed that *Zanthoxylum bungeanum* exhibits better affinity for H₂O₂ (Km = 27.5 mmol / L) compared to guaiacol. UPLC-MS / MS protein identification revealed four peroxidases with different molecular weights in the crude *Zanthoxylum bungeanum* enzyme. Their peptide sequences are similar to those found in rapeseed, mustard, lychee, and water celery, with molecular weights of 46.32, 39.41, 35.03, and 9.06 kDa, respectively.

[0091] Compared to commercially available horseradish peroxidase, Sichuan pepper exhibits better thermal and pH stability, with an optimal temperature of 50℃ and an optimal pH of 6.0. Its thermal deactivation mechanics conforms to a tandem inactivation model, with a high activation energy (299.65 kJ / mol) and strong thermal stability. When inhibiting peroxidase activity in the actual production and processing of Sichuan pepper, controlling the blanching temperature above 80℃ and the pH below 3 can effectively inhibit the peroxidase activity. Furthermore, common enzyme preparations such as ascorbic acid, salicylic acid, and citric acid can also effectively inhibit the peroxidase activity of Sichuan pepper.

[0092] 5. The effect of peroxidase on phenol removal and detoxification in bamboo leaf pepper.

[0093] To investigate the comprehensive utilization value of the bamboo leaf pepper peroxidase extracted by this method, the preliminarily purified bamboo leaf pepper peroxidase was used as a catalyst to combine with H2O2 to form a bamboo leaf pepper peroxidase-H2O2 system for the degradation of phenol wastewater, and the effect of this system on the catalytic degradation of phenol concentration under different conditions was investigated.

[0094] 5.1 Determination of Phenol Concentration

[0095] The phenol concentration during the reaction was determined colorimetrically: potassium ferricyanide (83.44 mmol / L) and 4-aminotipyrine (20.8 mmol / L) were prepared using 0.25 mol / L sodium bicarbonate solution. 100 μL of phenol solutions of different concentrations were taken, 4.85 mL of water was added, and the mixture was stirred. Then, 25 μL of potassium ferricyanide and 4-aminotipyrine were added respectively. The mixture was stirred thoroughly, and the absorbance was measured at 510 nm after a few minutes. A linear fit was performed with the absorbance at 510 nm as the Y-axis and the phenol concentration as the x-axis, yielding the relationship: Y5 = 0.1719X5 + 0.0739; R 2 =0.9995 (Y5: absorbance, X5: phenol concentration mmol / L), indicating a good linear relationship.

[0096] 5.2 Enzyme Concentration Assessment

[0097] This invention also investigated the effect of peroxidase concentration on phenol removal. The total reaction system was 10 mL. Phenol solution and buffer solution were first added to the 10 mL reaction system. The concentrations of H₂O₂ and phenol were fixed at 10 mmol / L. The enzyme concentrations in the reaction system were adjusted to achieve peroxidase activities of 20 U / mL, 40 U / mL, 80 U / mL, 200 U / mL, 280 U / mL, and 400 U / mL, and peroxidase concentrations of 80 U / mL, 200 U / mL, 400 U / mL, 560 U / mL, 800 U / mL, and 1600 U / mL, respectively. The effect of different peroxidase concentrations on phenol removal was investigated, and the results are as follows: Figure 9 .

[0098] The scavenging effect of *Zanthoxylum bungeanum* on phenol was investigated when the peroxidase activity in the reaction system was 20 U / mL, 40 U / mL, 80 U / mL, 200 U / mL, 280 U / mL, and 400 U / mL. Figure 9 A indicates that the degradation efficiency of phenol by peroxidase from *Zanthoxylum bungeanum* after three-phase extraction increases with increasing enzyme activity in the reaction system. However, the rate of change of the maximum removal rate gradually decreases as the peroxidase activity continues to increase. When the enzyme activity is 280 U / mL, the removal rate is 60.41%, while when the enzyme activity in the reaction system increases to 400 U / mL, the phenol removal rate remains at 60.66%. This shows that initially increasing the enzyme dosage can improve the phenol removal rate, but when the enzyme dosage reaches saturation in the reaction system, the catalytic efficiency decreases. Figure 9 A indicates that the peroxidase in *Zanthoxylum bungeanum* exhibits high phenol removal efficiency, reaching its maximum removal rate within 20 minutes, suggesting a rapid phenol removal reaction by the peroxidase in *Zanthoxylum bungeanum*. Figure 9 As shown in B, at all concentrations, horseradish peroxidase has a weaker phenol scavenging rate than bamboo leaf pepper peroxidase, and the best phenol scavenging rate is 37.97% when the peroxidase concentration is 800 U / mL.

[0099] 5.3 Investigation of Hydrogen Peroxide Concentration

[0100] After determining the optimal enzyme concentration, the concentration of hydrogen peroxide solution in the reaction system was adjusted to 1 mmol / L, 5 mmol / L, 10 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, and 40 mmol / L, respectively, to investigate the effect of hydrogen peroxide concentration on phenol removal. The results are shown below. Figure 10With increasing hydrogen peroxide concentration, the phenol clearance by *Zanthoxylum bungeanum* peroxidase initially increased and then decreased, reaching a maximum clearance of 69.46% at a hydrogen peroxide concentration of 20 mmol / L. Horseradish peroxidase showed the highest phenol clearance rate at 5 mmol / L, at 32.46%. Studies have shown that high concentrations of hydrogen peroxide inactivate peroxidase, hindering the reaction. Similarly, this study found that the phenol clearance rate of *Zanthoxylum bungeanum* peroxidase gradually decreased when the H2O2 concentration was greater than 20 mmol / L, and the phenol clearance rate of horseradish peroxidase gradually decreased when the H2O2 concentration was greater than 5 mmol / L.

[0101] 5.4 Effect of pH on phenol removal efficiency

[0102] To understand the application range of peroxidase from *Zanthoxylum bungeanum*, the effect of pH on the phenol removal efficiency of peroxidase from *Zanthoxylum bungeanum* was investigated, and compared with that of horseradish peroxidase. Based on pH 5.2–5.3, the enzyme dosage and hydrogen peroxide concentration were fixed. The pH of the reaction system was adjusted to 2.0–10.0 using 0.1 mol / L buffer solutions. Glycine hydrochloride buffer solution was used for pH 2.0–3.0, acetate buffer solution for pH 4.0–5.0, phosphate buffer solution for pH 6.0–8.0, and tris(hydroxymethyl)aminomethane hydrochloride buffer solution for pH 9.0–10.0. The effects of peroxidase on phenol removal at different pH values ​​are shown in the figure. Figure 11 .Depend on Figure 11 It was found that under different pH conditions, the scavenging effect of *Zanthoxylum bungeanum* peroxidase on phenol was stronger than that of horseradish peroxidase. At pH 8.0, the scavenging rate of *Zanthoxylum bungeanum* peroxidase reached its highest level of 75.60%, while horseradish peroxidase showed the best scavenging effect of 51.36% at pH 6.0. When the pH range was 3.0–8.0, the scavenging rate of *Zanthoxylum bungeanum* peroxidase ranged from 50.04% to 75.60%, while that of horseradish peroxidase ranged from 12.80% to 51.36%. The results indicate that *Zanthoxylum bungeanum* can adapt to a wider range of pH values, and its pH adaptation range is wider than that of horseradish peroxidase.

[0103] 5.5 Effect of Temperature on Phenol Removal

[0104] Temperature is a key factor affecting the phenol removal efficiency of peroxidase. Based on points 5.2–5.4, the optimal enzyme dosage, peroxide concentration, and pH value were selected to investigate the effect of temperature on phenol removal. The enzyme solution and phenol solution were incubated at 20–60°C for 30 min, then hydrogen peroxide was added to initiate the reaction. Samples were taken at 5 min, 20 min, 60 min, 120 min, and 180 min to determine the phenol concentration. The results of the effect of temperature on the phenol removal efficiency of peroxidase are shown below. Figure 12 .Depend on Figure 12 A indicates that when the temperature exceeds 50℃, the removal rate of phenol by peroxidase in *Zanthoxylum bungeanum* decreases rapidly, reaching only 40.01% after 3 hours. *Zanthoxylum bungeanum* peroxidase is stable between 30 and 60℃, but the phenol removal rate decreases at 60℃, likely due to the decomposition of H₂O₂ caused by high temperatures. After treatment at 30–50℃ for 3 hours, the phenol removal rate by *Zanthoxylum bungeanum* peroxidase ranged from 61.61% to 82.20%, with the optimal removal effect observed at 30℃. Figure 12 B indicates that horseradish peroxidase's phenol scavenging rate at 30–60℃ is 37.28%–41.33%, similar to that of bamboo leaf and Sichuan pepper peroxidase, showing better phenol scavenging effect at 30℃. Figure 12 It can be seen that the peroxidase reaction is rapid. In the initial 1 hour, the peroxidase of bamboo leaf pepper clears phenol at a relatively fast rate, but the rate gradually slows down in the later stage.

[0105] 5.6 Effect of phenol concentration on phenol removal efficiency

[0106] To determine the environmental application conditions of peroxidase from *Zanthoxylum bungeanum*, the scavenging rate of peroxidase against phenol was investigated at phenol concentrations ranging from 1 to 10 mmol / L. The scavenging rates of *Zanthoxylum bungeanum* at different phenol concentrations are shown in the following figures. Figure 13 .Depend on Figure 13 It was found that the scavenging rate of phenol by *Zanthoxylum bungeanum* peroxidase was above 82.40% within the phenol concentration range of 1–10 mmol / L. Horseradish peroxidase (HRP) showed good scavenging effect on low concentrations of phenol, but its scavenging effect was poor at higher phenol concentrations. When the phenol concentration was below 1 mmol / L, horseradish peroxidase could achieve 100% scavenging, but when the phenol concentration was 3–10 mmol / L, the scavenging rate of horseradish peroxidase was only 31.76%–48.95%, which was relatively low. The actual scavenging effect of *Zanthoxylum bungeanum* peroxidase on phenol is as follows: Figure 14 As shown, compared to horseradish peroxidase, phenol water produced more precipitate after treatment with Sichuan pepper peroxidase. In summary, Sichuan pepper peroxidase exhibits good scavenging rates at high, medium, and low concentrations of phenol, and its application range is wider than that of horseradish peroxidase.

[0107] 5.7 The detoxifying effect of peroxidase from bamboo leaf pepper on phenol water

[0108] The detoxification effect of Zanthoxylum bungeanum peroxidase (ZADTP) obtained by three-phase extraction on phenol water was evaluated using mung bean seeds. First, the seeds were surface-sterilized with 0.1% sodium hypochlorite, then washed five times with distilled water and placed in petri dishes lined with filter paper. Then, different solutions were added to the petri dishes: 5 mL of phenol solution (5 mM, 10 mM) and phenol solution treated with ZADTP and commercial horseradish peroxidase (HRP) were used as treatment groups (adjusted according to the highest scavenging enzyme activities screened in 5.2, so that the ZADTP activity was 200 U / mL and the commercial horseradish peroxidase activity was 280 U / mL), and distilled water was used as the control group. Seeds were germinated in the dark at 26°C, with three replicates for each treatment and control, and ten seeds per replicate. On the third day, the root and stem growth lengths of the seeds were recorded and compared. The results are shown below. Figure 15 As shown. Compared with the phenol water group (5mM Phenol: 3.56±0.16cm, 10mM: 2.46±0.12cm), the stem length of seedlings treated with phenol water and ZADTP was significantly increased (5mM Phenol + ZADTP: 4.67±0.17cm, 10mM Phenol + ZADTP: 3.50±0.14cm), while there was no statistically significant difference in stem length between the 5mM phenol water treatment and the control (4.67±0.10cm). Seedlings treated with HRP showed no significant growth compared with the control group (5mM Phenol + HRP: 3.17±0.01cm, 10mM Phenol + HRP: 2.48±0.10cm). Figure 15 (A, B). Similarly, the root length of ZADTP seedlings treated with different concentrations of phenol water (5 mg / mL phenol + ZADTP: 3.92 ± 0.17 cm; 10 mg / mL phenol + ZADTP: 3.51 ± 0.19 cm) was comparable to the control (3.90 ± 0.20 cm), significantly higher than that treated with untreated phenol water (5 mg / mL phenol: 2.19 ± 0.13 cm; 10 mg / mL phenol: 1.92 ± 0.10 cm), while HRP treatment showed no significant root growth (5 mg / mL phenol + HRP: 1.94 ± 0.12 cm; 10 mg / mL phenol + HRP: 1.68 ± 0.10 cm). Figure 15 C, D). Although the metabolites have not yet been identified, phytotoxicity assays indicate that ZADTP treatment produces non-toxic or less toxic metabolites compared to phenol water. Figure 15 E).

[0109] 5.8 Summary

[0110] This study compared the phenol removal capabilities of *Zanthoxylum bungeanum* peroxidase and commercial horseradish peroxidase. Compared to horseradish peroxidase, *Zanthoxylum bungeanum* peroxidase exhibited higher phenol removal efficiency, demonstrating better phenol removal rates over a shorter time and a wider pH and temperature range. This avoids the need for pre-adjustment of pH and temperature in industrial wastewater treatment, saving treatment time and reducing costs. Compared to commercial peroxidases, *Zanthoxylum bungeanum* peroxidase achieved better phenol removal rates even at lower enzyme activity and purity. Furthermore, the reduced toxicity of phenol-treated water and its promotion of plant growth after treatment with *Zanthoxylum bungeanum* peroxidase indicated that phenol-treated water with low-purity peroxidase degradation could be used in agricultural production, demonstrating the enzyme's promising potential. In addition, fresh *Zanthoxylum bungeanum* is inexpensive, and the extraction of *Zanthoxylum bungeanum* peroxidase is simple, requiring no complex equipment, further enhancing the feasibility of its industrial and environmental applications.

Claims

1. A method for removing phenolic substances from wastewater, characterized in that: Add bamboo leaf and Sichuan pepper peroxidase and hydrogen peroxide to wastewater containing phenolic substances; The phenolic substance is phenol; The peroxidase of *Zanthoxylum bungeanum* was obtained by extracting *Zanthoxylum bungeanum* with 10 times the amount of 0.05 mol / L phosphate buffer solution, adding 15% ammonium sulfate to the extract until saturated, and then performing three-phase extraction on the supernatant. The middle layer of the extract was dialyzed and freeze-dried to obtain the solid. The three-phase extraction process involves adding 45% ammonium sulfate to the supernatant until saturated, then adding tert-butanol, centrifuging, and obtaining the extract. The peroxidase from *Zanthoxylum bungeanum* contains four proteins with molecular weights of 46.32, 39.41, 35.03, and 9.06 kDa, respectively. The final concentration of the added bamboo leaf pepper peroxidase is 200~300 U / mL; The wastewater treatment process involves a pH range of 3.0 to 8.0 and a temperature range of 30 to 50°C.

2. The method according to claim 1, characterized in that, The final concentration of the peroxidase in the bamboo leaf pepper was 280 U / mL.

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