Peroxidase as well as extraction method and application thereof

Abstract

The invention provides a naturally extracted peroxidase which can be specifically extracted from at least one of leaves of abutilon indicum, leaves of livistona chinensis, leaves of abelmoschus moschatus and fruits of palm, and enzyme activity, protein content, specific activity, isoelectric point, relative molecular mass and the like are further explored, which shows that the thermal stability of the peroxidase is better than that of the existing peroxidase. Meanwhile, the invention further provides an extraction method of the peroxidase, the extraction method specifically comprises the steps of crude extraction, purification, freezing and drying, detailed operation processes and parameters in all the steps are specifically provided, and test data show that the extraction method is simple, efficient, large in yield, high in quality and easy to apply and popularize. Meanwhile, the invention also protects the specific application of the peroxidase in guaiacol-containing materials, especially pulp for preparing paper.

Description

Technical Field

[0001] The present application relates to the technical field of biological enzyme extraction, and in particular to a method for extracting biological enzymes from pure plants and its application. Background Art

[0002] Peroxidase (POD, EC1.11.1.-) is a class of oxidoreductases that catalyze the oxidation of phenols and their derivatives using H2O2 as an electron acceptor. They are widely found in organisms such as animals, plants, fungi, bacteria and algae. Typical PODs contain a ferrous protoporphyrin (heme) cofactor, catalyzing the oxidation of substrates while reducing H2O2 (or other peroxides) to water. This is a three-step catalytic cycle involving different enzyme intermediate forms, known as the Poulos-Kraut mechanism, as shown below:

[0003] P+ROOH→P(Ⅰ)+ROH+H2O

[0004] P(Ⅰ)+D+H + →P(Ⅱ)+D + ·

[0005] P(Ⅱ)+D+H + →P+D + ·

[0006] Where P represents the peroxidase molecule, ROOH is H2O2 or peroxide, and D is the substrate that acts as an electron donor and is oxidized to a highly reactive free radical D + ·, P(I) is formed at the active site, P(I) is reduced by one electron from the substrate molecule to produce compound P(II), and then a second one electron transfer is performed to return the enzyme to P. D + Further participate in various non-enzymatic reactions, including self-cross-linking reactions.

[0007] There are many systematic classifications of peroxidases. According to the presence or absence of a heme prosthetic group, they are initially divided into heme peroxidases and heme peroxidases. According to the information recorded in the KEGG database (https: / / www.genome.jp / kegg / ), non-heme peroxidases account for a small proportion, and most peroxidases carry a heme prosthetic group. Heme peroxidases are further divided into two superfamilies, namely the peroxidase-cyclooxygenase superfamily (PCOXS) and the peroxidase-catalase superfamily (PCATS). PCOXS is an animal peroxidase that participates in physiological reactions such as innate immunity and antioxidant defense reactions. PCATS is the most studied non-animal heme peroxidase superfamily, which is divided into three categories according to its source:

[0008] 1. Class I peroxidases from bacteria, such as cytochrome c peroxidase and ascorbate peroxidase, catalyze the conversion of superoxide anion radicals (·O2-) into O2 and H2O2, and are important enzymes in the intracellular antioxidant defense mechanism; 2. Class II peroxidases secreted by fungi, including MnP, LiP and VP, are involved in the oxidative degradation of lignin, etc.;

[0009] 3. Class III peroxidases of plant origin (unified number EC1.11.1.7) play a role in plant auxin metabolism, lignification, suberization, cross-linking of cell wall components, resistance to pathogens and aging.

[0010] In recent years, the application value of plant peroxidase in the fields of catalytic synthesis, sensor analysis, pollution control, etc. has been increasingly valued. Among them, HRP is the most deeply studied and widely used peroxidase, but its source is limited to crops such as horseradish that are only concentrated in Northeast my country, and its yield is limited. As early as 2018, the inventors of this application also reported that peroxidase was isolated from the fruit of Arecetriandra, and it was successfully applied to catalyze the oxidative polymerization of isoeugenol to enhance the anti-mildew properties of plant alkaloids. Al-Senaidy et al. studied that the optimum temperature of peroxidase extracted from date palm leaves was 55°C. Rodríguez et al. studied that the optimum temperature of peroxidase in African oil palm was 72.3°C, and the optimum catalytic temperature of HRP extracted from horseradish by Lavery et al. for guaiacol was 30°C. Obviously, palm-derived peroxidases are all mesophilic enzymes, which are more suitable for use in high-temperature working environments than HRP.

[0011] At present, there have been academic reports on the separation and purification of peroxidases with strong redox ability and good thermal stability from palm plants, but their development and utilization are still in the initial stage, and the extraction and purification processes are complex, with low extraction efficiency, poor extraction quality, and small unit extraction volume, which is completely inconvenient for widespread industrial application. Moreover, there is no data showing the application of peroxidase extracted from plants in pulp. How to make full use of the abandoned palm agricultural and forestry biomass resources in Guangdong, solve the practical problems existing in the pulp and paper industry, obtain systematic and simplified peroxidase extraction and characterization methods, and explore its wider application scenarios, is conducive to improving the scientific and technological content of the biomass economy, and is also of great reference significance for promoting the traditional manufacturing industry to move towards a green and sustainable development path. Summary of the invention

[0012] This application uses the common palm plants of the Guangdong region, such as royal palm, fan palm, and palm, as sources, and uses a systematic method of acetone precipitation, ion exchange chromatography, and ultrafiltration to extract and purify peroxidase from palm leaves and fruits, and explores and optimizes the properties and optimal reaction conditions of the peroxidase, including its optimal pH, optimal temperature, stability, inhibitors, reaction kinetic constants, inhibitors, stabilizers, etc., and uses gas chromatography-mass spectrometry to analyze the reaction products of peroxidase and guaiacol. In order to deeply understand the catalytic mechanism, a three-dimensional model of peroxidase is constructed using homology modeling technology, and the interaction sites and binding energies of the enzyme and ligands such as guaiacol are simulated and calculated to provide basic research data for the application of peroxidase. At the same time, a method and application of extracting the above-mentioned peroxidase with high efficiency and high quality are also proposed.

[0013] The specific technical solutions claimed in this application are as follows:

[0014] A peroxidase, wherein the peroxidase is extracted from a natural plant and satisfies at least one of the following conditions (1) to (8):

[0015] (1) The peroxidase activity ranged from 3.39 U / mL to 91.06 U / mL;

[0016] (2) the protein content of peroxidase ranged from 0.405 to 0.736 mg / mL;

[0017] (3) The specific activity of peroxidase ranged from 8.46 to 122.38 U / mg;

[0018] (4) The isoelectric point of peroxidase is less than 7;

[0019] (5) Relative molecular mass between 39 kDa and 50 kDa;

[0020] (6) The natural plant is at least one of royal palm leaves, fan palm leaves, soft-leafed thorny palm leaves, and palm fruit; when the natural plants are royal palm leaves, palm fruit, and soft-leafed thorny palm leaves, the order of the fat index of the peroxidase extracted from each of them is: palm fruit > royal palm leaves > soft-leafed thorny palm leaves;

[0021] (7) With guaiacol as substrate, when the concentrations of FeCl3, MnCl2, ZnCl2, and FeSO4 were all 5 mmol / L, Mn 2+ , Fe 3+ 、Zn 2+ , Fe 2+ Ions have an inhibitory effect on the activity of peroxidase, and the order of inhibitory intensity is: Fe 2+ >Mn2+ >Fe 3+ >Zn 2+ ;

[0022] (8) 1 g / L Span-80 surfactant can increase the enzymatic activity of the peroxidase by at least 12%.

[0023] As a further improvement of the above scheme, the total activities of peroxidase obtained from every 100 g of Livistona leaves, Eryngium leaves and palm fruits were 20.33, 36.45 and 3.69 kU, respectively.

[0024] As a further improvement to the above scheme, the optimum pH of the peroxidase extracted from the leaves of Eryngium serrata is 4, and the optimum pH of the peroxidase extracted from the leaves of royal palm, the leaves of Livistona chinensis, and the fruits of palm are all 5.

[0025] As a further improvement to the above scheme, when the pH is between 3 and 6, the relative enzyme activities of the leaves of the fan palm, the leaves of the soft-leaved thorn palm and the palm fruit are all above 60%.

[0026] As a further improvement to the above scheme, the optimum catalytic temperature of the peroxidase is not less than 70°C.

[0027] As a further improvement to the above solution, the peroxidase can maintain more than 60% of its enzyme activity at a temperature of 90°C.

[0028] The present application also provides a method for extracting peroxidase as described in any of the previous technical solutions, comprising the following steps:

[0029] A. Obtaining crude peroxidase solution

[0030] The natural plant is collected to extract peroxidase, and the process includes:

[0031] Wash with deionized water and drain; weigh 100g of leaves or fruits, cut the leaves to less than 2cm, mix with 150mL of phosphate buffer solution with a concentration of 0.01mol / L and pH 5, put into a plant wall breaking machine for high-speed wall breaking for 1min to obtain a homogenate, then filter with 8 layers of absorbent cotton gauze, and then centrifuge for 10min to remove the filter residue to obtain a clear liquid, which is used as the initial liquid for subsequent separation and extraction, recorded as water extract; slowly drip the water extract into a certain volume of acetone, stir while dripping, stand at 4℃ for 15min, centrifuge for 10min to remove the supernatant, obtain a precipitate, dissolve the precipitate with phosphate buffer, make the volume to 100mL, centrifuge again to remove insoluble matter, and obtain a crude enzyme solution;

[0032] B. Purification of crude peroxidase solution

[0033] The crude enzyme solution obtained by acetone extraction is pumped into an ion exchange chromatography column to remove pigments and impurities, specifically including:

[0034] (1) Ion exchanger pretreatment: DEAE cellulose-DE52 was soaked in deionized water for 3 h, soaked in 0.5 mol / L NaOH solution for 30 min, then washed with deionized water until neutral, and then soaked in 0.5 mol / L HCl solution for 30 min and washed again until neutral;

[0035] (2) Column loading: The pretreated DEAE cellulose-DE52 is loaded into the chromatography column, and the chromatography column is fully equilibrated with a 0.01 mol / L phosphate buffer solution with a pH of 5;

[0036] (3) Sample loading: 7 mL of crude enzyme solution was added and balanced with 15 mL of phosphate buffer solution;

[0037] (4) Elution and collection: During elution, 2 mol / L NaCl solution and phosphate buffer solution were used for gradient mixed elution. One tube of sample was collected every 5 minutes. The elution time was 2 hours, and a total of 24 tubes of samples were collected. The peroxidase activity and protein concentration of each tube of sample were detected.

[0038] (5) Regeneration of the exchanger: Use 1 mol / L HCl and 1 mol / L NaOH for transformation regeneration. If the regenerated ion exchanger is not used for a long time, store it at 4°C to prevent microbial growth;

[0039] C. Desalting and freeze drying of peroxidase

[0040] The enzyme solution after ion exchange chromatography is desalted with an ultrafiltration centrifuge tube, the enzyme solution is placed in the ultrafiltration centrifuge tube, centrifuged at 5000rpm for 10 minutes, the enzyme trapped in the centrifuge tube is dissolved with deionized water, centrifuged again, and the above operation is repeated many times until Cl- is not detected in the centrifuge liquid passing through the filter membrane; finally, the trapped enzyme is dissolved in phosphate buffer, and freeze-dried into powder with a vacuum freeze dryer to obtain peroxidase.

[0041] As a further improvement to the above scheme, the peroxidase extraction method further satisfies at least one of the following conditions:

[0042] (1) In step A, the centrifugation condition is 4°C, 10000 rpm;

[0043] (2) The diameter of the chromatography column in step B is 1.6 cm and the length is 30 cm.

[0044] The present application also relates to an application of the peroxidase described in any of the previous technical solutions in catalysis or oxidation in a material containing guaiacol.

[0045] As a further improvement to the above solution, the guaiacol-containing material is pulp for preparing paper.

[0046] Beneficial technical effects

[0047] The invention uses royal palm, fan palm and palm, which are common palm plants in Guangdong area, as sources, and comprehensively uses a systematic method of acetone precipitation, ion exchange chromatography and ultrafiltration to extract and purify peroxidase from palm leaves and fruits, and determines the optimal pH, optimal temperature, stability, inhibitor, reaction kinetic constant, inhibitor, stabilizer and other enzymatic properties of the peroxidase, and uses a gas chromatography-mass spectrometer (GC-MS) to analyze the reaction products of the peroxidase and guaiacol.

[0048] Peroxidases were extracted from king coconut leaves, palm leaves, soft-leaved thorn palm leaves (PRLP) and palm fruits, and purified by acetone precipitation, ion exchange chromatography and ultrafiltration. Their molecular weight and kinetic constants were determined. The peroxidases obtained were acidophilic (pH 4-5), mesophilic (60℃-70℃) enzymes with good thermal stability. Common metal ions (Mn 2+ , Fe 3+ 、Zn 2 + The inhibitory effects of organic solvents (formaldehyde, ethanol, methanol, etc.), and ionic surfactants on peroxidase are studied. Nonionic surfactants can be used as stabilizers or promoters for enzymes. Through the optimization and screening of the above extraction, purification, ultrafiltration, temperature, metal ion inhibition, organic solvent inhibition, etc., an efficient, high-quality, simple extraction method and the extracted peroxidase are obtained, which provides a guarantee for the high-yield production and wide application of peroxidase extracted from natural plants.

[0049] The gas chromatography-mass spectrometry (GC-MS) was used to determine that the products of PRLP-catalyzed guaiacol oxidation were mainly dimers and trimers covalently linked by benzene ring CC. The effects of hydrogen bonds and hydrophobic forces between guaiacol monomers and peroxidase were studied by computer simulation, and it was predicted that peroxidase could accommodate guaiacol polymerization products with a degree of polymerization of 10 to enter the active center for oxidation reaction. The larger the degree of polymerization, the more stable the binding. This provides a theoretical basis and technical support for the application of peroxidase oxidation of guaiacol.

[0050] In addition, in order to gain a deeper understanding of the oxidative catalytic mechanism of natural peroxidase extracted from plants, a three-dimensional model of peroxidase was constructed using homology modeling technology, and the interaction sites and binding energies between the enzyme and ligands such as guaiacol were simulated and calculated, providing basic research data and support for further, broader and deeper applications of peroxidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a flow chart for the separation and purification of peroxidase;

[0052] Figure 2 The elution curve of peroxidase DEAE cellulose DE-52 chromatography, including (a) RRLP; (b) LCLP; (c) PRLP; (d) TFFP;

[0053] Figure 3 Zeta potential diagrams of four peroxidases at different pH values, including: (a) RRLP; (b) LCLP; (c) PRLP; (d) TFFP;

[0054] Figure 4 The results of the optimal conditions experiment of four kinds of peroxidases are shown in Figure 1, including (a) the optimal pH; and (b) the optimal temperature;

[0055] Figure 5 : Effects of storage temperature and time on the activity of four peroxidases, including (a) RRLP; (b) LCLP; (c) PRLP; (d) TFFP;

[0056] Figure 6 Linewear-Burk curves of peroxidase-catalyzed substrate oxidation, including (a) four enzymes for guaiacol substrate; (b) PRLP for three substrates;

[0057] Figure 7 The test results of the effects of common metal ions on peroxidase activity are shown in Figure 1, including (a) RRLP; (b) LCLP; (c) PRLP; (d) TFFP;

[0058] Figure 8 The test results of the effects of common organic solvents on peroxidase activity are shown in Figure 1, including (a) RRLP; (b) LCLP; (c) PRLP; (d) TFFP;

[0059] Fig. 9 The test results of the effects of common surfactants on peroxidase activity are shown in Figure 2, including (a) RRLP; (b) LCLP; (c) PRLP; (d) TFFP;

[0060] Fig.10 is the GC-MS graph of the guaiacol oxidation product by PRLP / H2O2;

[0061] Fig.11 This is the mechanism diagram of guaiacol polymerization catalyzed by PRLP / H2O2;

[0062] Fig.12The 20 conformations of peroxidase docked with guaiacol, including (a) RRLP; (b) TFFP; (c) PRLP;

[0063] Fig.13 This is a computer simulation of the binding energy results of docking guaiacol polymers with different polymerization degrees with peroxidase;

[0064] Fig.14 It is the protein ribbon diagram, where: (a) PRLP; (b) 4cuo; (c) PRLPvs4cuo;

[0065] Fig.15 Evaluation diagrams of the SAVES model before and after 3DRefine optimization: (a, b) ERRAT analysis diagram; (c, d) RamachandranPlot analysis diagram. DETAILED DESCRIPTION

[0066] The technical solutions of the specific embodiments of the present invention will be clearly and completely described below in combination with various specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, can be obtained through ordinary commercial channels.

[0067] Description of experimental reagents

[0068] Guaiacol (analytical grade) was purchased from Shanghai McLean Biotechnology Co., Ltd.; hydrogen peroxide (30%) was purchased from Guangzhou Chemical Reagent Factory; Coomassie Brilliant Blue G-250 (analytical grade) and bovine serum albumin (analytical grade) were purchased from Solarbio; DEAE cellulose-DE52 was purchased from Whatman; SDS-PAGE electrophoresis kit (BL508A) was purchased from Biosharp; other reagents were analytical grade. Mature palm leaves and fruits were picked from the Wushan Campus of South China University of Technology (113.344°E, 23.153°N) in September 2022 and were purified according to the attached Figure 1 Flow chart for extraction of peroxidase is shown.

[0069] The present application claims a peroxidase extracted from natural plants and a method for extracting the same. The technical solution is as follows:

[0070] The extraction method includes:

[0071] A. Obtaining crude peroxidase solution

[0072] Mature leaves or fruits of palm plants were collected, specifically: royal palm (Roystonearegia) leaves, fan palm (Livistonachinensis) leaves, soft-leaved thorny palm (Phoenixroebelenii) leaves, and palm (Trachycarpusfortunei) fruits, and peroxidase was extracted. The extracted products were recorded as: RRLP, LCLP, PRLP, TFFP (the finished product numbers listed later all correspond to the finished products extracted from different raw materials here).

[0073] The specific extraction process is:

[0074] Wash with deionized water and drain. Weigh 100g of leaves (cut to less than 2cm) or fruits, mix with 150mL phosphate buffer solution (0.01mol / L, pH5), put into plant wall breaking machine (FW80, Tianjin Test) at high speed (10000rpm) for 1min to obtain homogenate, then filter with 8 layers of absorbent cotton gauze, and centrifuge (AG-22331, Germany Eppendorf) (4℃, 10000rpm) for 10min to remove the filter residue and obtain clear liquid, which is used as the initial liquid for subsequent separation and extraction, recorded as water extract. The water extract was slowly dripped into a certain volume of acetone (water extract: acetone = 1:1, corresponding to an acetone volume fraction of 50vt%), with stirring while dripping. After standing at 4°C for 15 min, the supernatant was removed by centrifugation (4°C, 10000rpm) for 10 min to obtain a precipitate. The precipitate was dissolved with phosphate buffer, the volume was fixed to 100 mL, and the insoluble matter was removed by centrifugation again to obtain a crude enzyme solution.

[0075] B. Purification of crude peroxidase solution

[0076] The crude enzyme solution obtained by acetone extraction is pumped into an ion exchange chromatography column to remove pigments and impurities. The steps are as follows:

[0077] (1) Ion exchanger pretreatment: DEAE cellulose-DE52 was pre-soaked in deionized water for 3 h to swell, soaked in NaOH solution (0.5 mol / L) for 30 min, then washed with deionized water until neutral, and then soaked in HCl solution (0.5 mol / L) for 30 min and washed again until neutral.

[0078] (2) Column loading: The pretreated DEAE cellulose-DE52 was loaded into a chromatography column (1.6 cm in diameter, 30 cm in length), and the chromatography column was fully equilibrated with a phosphate buffer solution (0.01 mol / L, pH 5).

[0079] (3) Sample loading: Add 7 mL of crude enzyme solution and balance with 15 mL of phosphate buffer solution.

[0080] (4) Elution and collection: NaCl solution (2 mol / L) and phosphate buffer solution were used for gradient elution. One tube of sample was collected every 5 minutes. The elution time was 2 hours, and a total of 24 tubes of samples were collected. The peroxidase activity and protein concentration of each tube of sample were tested.

[0081] (5) Regeneration of the exchanger: Use 1 mol / L HCl and 1 mol / L NaOH for transformation regeneration. If the regenerated ion exchanger is not used for a long time, store it at 4°C to prevent microbial growth.

[0082] C. Desalting and freeze drying of peroxidase

[0083] The enzyme solution after ion exchange chromatography was desalted using an ultrafiltration centrifuge tube (MWCO 10 kDa, Millipore, USA). The enzyme solution was placed in an ultrafiltration centrifuge tube and centrifuged at 5000 rpm for 10 min using a centrifuge (SC-3610, Anhui Zhongjia). The enzyme trapped in the centrifuge tube was dissolved in deionized water and centrifuged again. The above operation was repeated several times until no Cl was detected in the centrifuge solution passing through the filter membrane. - Finally, the retained enzyme was dissolved in phosphate buffer and freeze-dried into powder using a vacuum freeze dryer (Alpha1-2LDplus, Christ, Germany) to obtain peroxidase.

[0084] Performance Testing

[0085] Determination of peroxidase activity and protein content

[0086] The activity of peroxidase was determined by the guaiacol method. In the presence of trace amounts of H2O2, peroxidase catalyzes the oxidation of guaiacol to produce brown-red oxidation products with an absorption peak at 470nm. The enzyme activity (U) is defined as the amount of enzyme required for the absorbance value at 470nm to change by 0.01 per 1min. In the enzyme activity test, 2.7mL of phosphate buffer solution (0.01mol / L, pH5), 0.1mL of H2O2 (0.1mol / L) and 0.1mL of guaiacol solution (0.1mol / L) were measured and added to a 1cm×4cm quartz cuvette. Finally, 0.1mL of enzyme solution was added to start the reaction. The change in absorbance value at 470nm wavelength within 3min was measured using an ultraviolet-visible spectrophotometer (UV-1900, Shimadzu, Japan), and the peroxidase activity was calculated according to the following calculation formula.

[0087]

[0088] Where ΔA 470 ——absorbance change value; V——reaction enzyme solution volume, mL; t——reaction time, min.

[0089] The protein content of peroxidase was determined by Coomassie Brilliant Blue G-250 staining method, with bovine serum albumin (BSA) as a standard. The preparation method of Coomassie Brilliant Blue G-250 working solution is as follows: 120 mg Coomassie Brilliant Blue G-250 solid was dissolved in 50 mL ethanol (95%), 100 mL concentrated phosphoric acid (85%) was added, and then the volume was made up to 1 L, stored in the dark, and filtered with filter paper before use. The preparation of BSA standard curve was as follows: 30 μL BSA standard solution of different concentrations was added to a test tube, and 3 mL Coomassie Brilliant Blue G-250 working solution was added and mixed evenly, and the solution was kept in the dark for 5 min at room temperature, and the absorbance value at 595 nm was measured by UV-Vis spectrophotometer. The difference between the absorbance of the standard sample and the blank sample (water instead of BSA standard solution) is taken as the ordinate (y), and the BSA concentration is taken as the abscissa (x). The equation of the standard curve is: y = 0.3066x, R 2 =0.9970, n=8.

[0090] When testing the sample, add 30 μL of sample to replace the BSA standard solution, obtain the absorbance value, and calculate the protein content using the standard curve equation.

[0091] The specific activity (U / mg) of an enzyme is defined as the ratio of enzyme activity (U / mL) to protein content (mg / mL). Generally speaking, for enzymes from the same source but with different degrees of purification, the greater the specific activity value, the higher the purity.

[0092] First, the protein content and enzyme activity of the water extract (calculated based on 100g of leaves or fruits) were determined, and the results are shown in Table 1. The water extract of sample LCLP had the highest enzyme activity, which was 85.88U / mL; PRLP and RRLP were second, with enzyme activities of 35.64U / mL and 35.09U / mL, respectively. The enzyme activity of TFFP water extract was the lowest, only 3.56U / mL. In terms of protein content, the water extracts of these enzymes were not much different, with the highest being 0.734mg / mL for LCLP and the lowest being 0.406mg / mL for TFFP. LCLP had the highest specific activity, which was 116.9U / mg, followed by PRLP with a specific activity of 72.71U / mg.

[0093] Table 1 Summary of protein content and enzyme activity of peroxidase water extract

[0094] Water extract Enzyme activity (U / mL) Protein content (mg / mL) specific activity (U / mg) RRLP 35.09±4.68 0.608±0.0157.60±9.55 LCLP 85.88±5.18 0.734±0.02116.90±5.48 PRLP 35.64±4.77 0.489±0.0172.71±11.69 TFFP 3.56±0.17 0.406±0.018.76±0.29

[0095] Note: Each experiment was performed in triplicate and the average value was taken. The error was the standard deviation.

[0096] See the instruction manual Figure 2 , is the elution curve of each peroxidase sample. Among them, Figure 2 (a) is the elution curve of RRLP. The elution curve has three protein peaks at 35min, 60min and 100min. Only one enzyme activity peak was detected, which appeared at 35min, close to the first protein peak. No enzyme activity was detected near the second and third elution peaks, indicating that these two peaks are impurities. Figure 2 (a) It also reflects that rapid liquid chromatography can effectively remove impurities. The eluate corresponding to the enzyme activity peak was collected, and the specific enzyme activity was 1477.73U / mg, which was 25.65 times purified compared with the water extract. It was calculated that the activity of the purified peroxidase obtained from every 100g of royal palm leaves was 21.1kU. However, if calculated from Table 1, the total peroxidase activity per 100g of royal palm leaves is only 3.5kU. The reason is probably that the leaves contain a large amount of antioxidant ingredients such as anthocyanins, which inhibit the activity of peroxidase in the water extract. After acetone extraction and ion exchange chromatography, these antioxidant ingredients were separated, so the peroxidase showed stronger activity.

[0097] Figure 2 (b), 2(c) and 2(d) are the elution curves of LCLP, PRLP and TFFP, respectively. Similar to the curve of RRLP, there are three protein peaks, and the enzyme activity peaks all appear at the first protein peak. The eluates corresponding to the enzyme activity peaks were collected, and the specific enzyme activities were measured to be 1139.16U / mg, 5103.47U / mg and 397.04U / mg, respectively. Compared with the water extract, they were purified by 9.74 times, 70.19 times and 44.51 times, respectively. That is, the total peroxidase activities obtained from 100g of palm leaves, soft-leaved thorn palm leaves and palm fruits were 20.33, 36.45 and 3.69kU, respectively.

[0098] Estimation of molecular weight of peroxidase

[0099] Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis was used to estimate the molecular weight of peroxidase. The specific steps are as follows:

[0100] (1) Pouring 10% separation gel: Take 3.3mL 30% acrylamide-methylenebisacrylamide solution (30% Acr-Bis), 3.8mL Tris-HCl buffer solution (1mol / L, pH8.8), 0.1mL 10% SDS and 2.7mL distilled water and mix them in a beaker, then add 0.01mL N,N,N',N'-tetramethyldiethylamine (TEMED) and 0.1mL 10% ammonium persulfate (10% APS). Stir slowly to mix evenly. Slowly add the separation gel into the glass tank to about 1.5cm from the top of the front glass plate, and cover the separation gel with a water layer of about 3mm to make the gel surface uniform and flat. Let it stand for 20 minutes. After the gel is polymerized, there is a clear interface between the separation gel and the water layer.

[0101] (2) Casting 4% stacking gel: Carefully pour off the water layer on the separation gel, then mix 0.33 mL 30% Acr-Bis, 0.25 mL Tris-HCl buffer solution (1 mol / L, pH 6.8), 0.02 mL 10% SDS and 1.4 mL distilled water, then add 0.002 mL TEMED and 0.02 mL 10% APS. After mixing the reagents evenly, add the stacking gel to the top of the separation gel, insert the comb into the gel, and pull it out after the stacking gel polymerizes.

[0102] (3) Sample preparation and loading: Mix the enzyme and loading buffer in a centrifuge tube and heat at 95°C for 10 min. Then, take 10 μL of protein sample and add it to the sample well of the stacking gel.

[0103] (4) Electrophoresis: Fix the gel plate in a vertical electrophoresis tank, connect the electrode plug of the electrophoresis instrument (Mini-PROTEANTetra, Bio-Rad, USA) to the power supply, and the current flows to the anode. When the sample moves in the concentrated gel, the voltage is adjusted to 70V. When the front edge of the sample is located at the interface of the concentrated gel and the separation gel, the voltage is adjusted to 160V. Turn off the power supply when the sample moves to the bottom of the gel.

[0104] (5) Coomassie Brilliant Blue Staining: Gently pry open the glass plate, place the gel in a container containing Coomassie Brilliant Blue staining solution, and slowly shake and stain for 30 minutes. Then pour out the staining solution, add methanol destaining solution, and replace the destaining solution every 2 hours. When clear bands appear, the destaining is complete. After pouring out the destaining solution, take out the gel and take a picture of the destaining gel using a chemiluminescent gel imaging system (Bio-Rad ChemiDoc XRS). The molecular weight of the sample is estimated by calculating the relative mobility of the sample and the marker.

[0105] The purified peroxidase was obtained after purification by acetone, ion exchange chromatography and desalting. Its purity was detected by SDS-PAGE electrophoresis. The results showed that all four peroxidase samples had only one protein band, which indicated that the purified enzyme had reached the electrophoretic purity level. By comparing the protein markers, the relative molecular masses of RRLP, LCLP, PRLP, and TFFP were calculated by mobility to be 39kDa, 44kDa, 50kDa and 48kDa, respectively.

[0106] Peroxidase isoelectric point (pI) determination

[0107] The pI of purified peroxidase was determined using a Zeta potential analyzer. First, 1 g / L peroxidase solution was prepared with deionized water. 0.1 mol / L HCl solution was added to adjust the initial pH of the enzyme solution to 2.5. Then, 0.1 mol / L NaOH solution was used for titration. The changes in pH and Zeta potential during the titration were recorded. The pH value when the Zeta potential was zero was the isoelectric point of the enzyme.

[0108] See the instruction manual Figure 3 Enzymes are amphoteric polyelectrolytes with unique charge properties in solution. When the net charge is zero, the pH is defined as the isoelectric point pI. This experiment uses the Zeta potential method to test pI. The Zeta potential of peroxidase changes with pH as shown in the figure. Figure 3 As shown, it can be seen that as the pH increases, the Zeta potential of the peroxidase gradually decreases. When the Zeta potential is 0, the pIs of RRLP, LCLP, PRLP and TFFP are 5.1, 5.5, 5.4 and 5.7 respectively. The above data show that the peroxidase prepared in this application is an acidic protein (pI<7).

[0109] Testing of optimal pH, optimal temperature and temperature stability

[0110] Dissolve the peroxidase in a phosphate buffer with a pH of 2 to 10 to a concentration of 1.0 g / L, test the enzyme activity and determine its optimal pH. Dissolve the peroxidase in a phosphate buffer with an optimal pH value, test the optimal temperature of the enzyme in the temperature range of 25 to 100°C, and take samples every 1 hour to test its temperature stability. Set the highest enzyme activity as 100%, and calculate the relative enzyme activity according to the following formula:

[0111]

[0112] Enzymes will only show high activity under suitable pH and temperature conditions. Using guaiacol as substrate, the activities of four peroxidases under different pH conditions were obtained. The results are as follows: Figure 4As shown in (a). Except for PRLP, whose optimum pH is 4, the optimum catalytic pH of the other three enzymes is 5. Between pH = 3 and 6, the relative enzyme activities of LCLP, PRLP and TFFP are all above 60%, and the relative activity of PRLP is about 50%. These enzymes are not alkali-resistant. When pH>7, the enzyme activity is lost rapidly and is almost completely inactivated under alkaline conditions. Generally speaking, the suitable pH value for an enzyme is also its stable pH value. The results of the optimum pH determination confirm that these four enzymes are acidic enzymes and are suitable for catalyzing the oxidation of guaiacol under acidic conditions. Commercial horseradish peroxidase (HRP, ) The optimum pH is 6.5. Compared with the above plant-derived peroxidases, the palm-derived peroxidase has better acidity. It should be pointed out that the optimum pH and pI of the peroxidase measured in this experiment are close, but there is no correlation between the two in enzymology. In subsequent innovative practices, unless otherwise specified, pH 5 is used for peroxidase activity testing.

[0113] The influence of temperature Figure 4 As shown in Figure (b), the optimum catalytic temperature of RRLP, LCLP, PRLP and TFFP all reached 70°C. All four enzymes showed good mesophilicity. When tested at 90°C, all four enzymes could maintain more than 60% of their enzyme activity. Obviously, the palm-derived peroxidases are all mesophilic enzymes, which are more suitable for use in high-temperature working environments than the peroxidases in the prior art.

[0114] At the same time, the inventors found through innovative practice that the stability of peroxidase activity was measured after storage at different temperatures for a period of time. Figure 5 . After RRLP was kept at 50℃ for 8h, the remaining enzyme activity was equivalent to 83% of the initial value; the higher the temperature, the more serious the storage inactivation, and the enzyme activity lost 44% after being kept at 60℃ for 8h. 70℃ is the optimal catalytic temperature for RRLP. When stored at 70℃, RRLP can be stable for 1h, during which the enzyme activity loss does not exceed 15%; but after being kept warm for 2h, the relative enzyme activity dropped to 42%; after being kept warm for 8h, RRLP completely lost its activity. This also shows that the optimal temperature for RRLP is actually a balance between the increase in the rate of enzyme catalytic reaction and the loss of enzyme activity due to thermal denaturation. The thermal stability of LCLP is significantly better than that of RRLP. Its inactivation is not significant after being kept warm at 70℃ for 2h, and there is still 50% of the residual enzyme activity after being kept warm for 8h, but LCLP cannot withstand the high temperature of 80℃.

[0115] PRLP showed better thermal stability than LCLP. When kept at 50℃ or 60℃ for 8h, the enzyme activity loss was less than 10%. After being kept at 80℃ for 8h, 40% of the enzyme activity was still retained. Even after being kept at 90℃ for 1h, the relative enzyme activity was still more than 40%. In contrast, LCLP and RRLP were completely inactivated after being kept at 90℃ for 1h. One of the important characteristics of PRLP is that it catalyzes oxidation reactions at high temperatures (80-90℃).

[0116] For the thermal stability test results of TFFP, see the attached manual. Figure 5 As shown in Figure (d), at 70°C, the half-life of TFFP exceeds 8 hours, and at 80°C, the half-life is about 1.5 hours.

[0117] Determination of Michaelis constant (Km)

[0118] The Michaelis-Menten equation (the formula below) reflects the relationship between the enzyme reaction rate and the substrate concentration. 1 g / L enzyme solution was reacted with different concentrations of guaiacol and H2O2, and the change rate of absorbance at 470 nm within 3 min was recorded to calculate the enzyme reaction rate (V, μmol·L-1·min-1). vmax and Km were calculated according to the Linewear-Burk double reciprocal formula.

[0119]

[0120] Vmax——maximum reaction rate, μmol·L-1·min-1; [s]——substrate concentration, mmol·L-1; Km——Michaelis constant, mmol·L-1.

[0121]

[0122] The Michaelis-Menten equation was used to analyze the relationship between the peroxidase catalytic reaction rate and the guaiacol substrate concentration, and the Linewear-Burk double reciprocal plot was obtained (see Figure 6 Figure a). It was calculated that at room temperature and pH 5, the Km values ​​of guaiacol oxidation catalyzed by four peroxidases, RRLP, LCLP, PRLP and TFFP, were 3.63mmol·L-1, 5.44mmol·L-1, 3.13mmol·L-1 and 2.58mmol·L-1, respectively. The Vmax values ​​were 0.043μmol·L-1·min-1, 0.018μmol·L-1·min-1, 0.030μmol·L-1·min-1 and 0.0020μmol·L-1·min-1, respectively. The affinity for guaiacol was TFFP>PRLP>RRLP>LCLP.

[0123] The same enzyme has different catalytic rates for different substrates. Figure 6 (b) in the figure), the inventors found that under the conditions of room temperature and pH 5, the Km of PRLP catalyzing guaiacol, catechol and gallic acid were 3.13mmol·L-1, 2.43mmol·L-1 and 2.18mmol·L-1, respectively, and the Vmax were 0.030μmol·L-1·min-1, 0.0075μmol·L-1·min-1 and 0.0095μmol·L-1·min-1, respectively. The affinity of PRLP to substrates is in the order of gallic acid>catechol>guaiacol. The Km of the same enzyme for different substrates is different, and the substrate with the smallest Km is usually called the most suitable substrate for the enzyme.

[0124] Effects of metal ions on peroxidase activity

[0125] Peroxidase itself is an enzyme that relies on metal ions for catalytic reactions. Some metal ions may also bind to key amino acid residues in the catalytic center of the enzyme, thereby inhibiting the activity of the enzyme. Therefore, this study tested the effects of several common metal ions on peroxidase activity. Including the effects of metal salts such as NaCl, KCl, CaCl2, FeCl3, MnCl2, ZnCl2, BaCl2, CuSO4, MgSO4 and FeSO4 on peroxidase activity. First, prepare phosphate buffer solutions (0.01mol / L, pH5) containing 5mmol / L and 10mmol / L metal salts, respectively, then dissolve the enzyme lyophilized powder, prepare an enzyme solution (containing metal salts) with a concentration of 1g / L, and determine the peroxidase activity according to the above method.

[0126] See the instruction manual Figure 7 The results showed that, with guaiacol as substrate, Na + , K + , Ca 2+ , Ba 2+ and Mg 2+ The effect on peroxidase activity is small. At a concentration of 10mmol / L, the enzyme activity is retained above 88%. 2+ , Fe 3+ 、Zn 2+ , Fe 2+ The ions showed significant inhibitory effects on the four enzymes, and the inhibition intensity was ranked as follows: Fe 2+ >Mn 2+ >Fe 3+ >Zn 2+ The peroxidases from different species are significantly affected by metal ions. 2+It is the strongest inhibitor among all the metal ions tested in this experiment. When the concentration reaches 10mmol / L, the remaining enzyme activities of RRLP, LCLP, PRLP and TFFP are only 3.64%, 1.80%, 6.37%, 2.14% and 8.45% respectively.

[0127] Effects of organic solvents on peroxidase

[0128] Organic solvents are often used in enzymatic reactions to increase the solubility of substrates or products, but the addition of organic solvents will destroy the three-dimensional structure of enzyme proteins, thereby inhibiting the activity of the enzyme. The present invention investigates the effects of organic solvents such as methanol, ethanol, acetone, formaldehyde, isopropanol, ethylene glycol and tert-butyl alcohol on the activity of peroxidase. Phosphate buffer solutions containing 15% (v / v) and 30% (v / v) organic solvents are prepared respectively, and then the enzyme freeze-dried powder is dissolved to prepare 1g / L enzyme solution (containing organic solvent), and the peroxidase activity is measured.

[0129] See the instruction manual Figure 8 , reflecting the effect of the investigated organic solvents on the activity of peroxidase. Among them, ethylene glycol has a smaller inhibitory effect on the enzyme. When the volume fraction is 15%, except for TFFP, which maintains 60% enzyme activity, RRLP, LCLP, and PRLP all have more than 70% residual enzyme activity. When the volume fraction is 30%, RRLP, LCLP, and PRLP still have 40-60% relative enzyme activity, and TFFP has 21% residual enzyme activity. In 15% acetone solution, RRLP, LCLP, PRLP, and TFFP have 23%, 28%, 33%, 19%, and 30% residual enzyme activity, respectively. When the volume fraction reaches 30%, the relative residual enzyme activity of the four enzymes is all below 10%. It should be pointed out that although the activity of the four peroxidases is strongly inhibited in 30% volume fraction acetone solution, this is not due to irreversible denaturation. Before purification by ion exchange chromatography, all peroxidases were separated by precipitation with 50% acetone, redissolved in water and passed through the exchange column. The enzyme activity peak could still be detected. This phenomenon shows that the denaturing effect of acetone on the enzyme is reversible.

[0130] When other solvents such as ethanol, methanol, isopropanol and tert-butyl alcohol were added to a volume fraction of 30%, the remaining relative enzyme activities of the four enzymes were all below 20%. These solvents are commonly used reversible protein precipitants, and their effects are likely similar to acetone. However, formaldehyde is different from these solvents. Figure 2-10 The data showed that no peroxidase activity was detected in all tests with the addition of formaldehyde. The four peroxidases were completely inactivated in formaldehyde. The reason may be that formaldehyde has a strong cross-linking effect on the enzyme, causing the enzyme to undergo irreversible precipitation and complete inactivation.

[0131] Effects of surfactants on peroxidase

[0132] The effects of surfactants such as cocamidopropyl betaine (CAB), cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), Tween-40, Tween-80 and Span-80 on peroxidase activity were investigated. Phosphate buffer solutions containing 1g / L and 10g / L surfactants were prepared respectively, and then the enzyme lyophilized powder was dissolved to prepare 1g / L enzyme solution (containing surfactant) to measure the peroxidase activity.

[0133] The effects of commonly used surfactants on peroxidase activity are shown in Fig. 9 Cationic surfactants

[0134] (such as CAB and CTAB) and anionic surfactants (SDS) are inhibitors of peroxidase, among which CTAB has a stronger inhibitory effect. At a concentration of 1g / L, the peroxidase activities of RRLP, LCLP, PRLP, and TFFP were lost by 56%, 52%, 41%, 61%, and 56%, respectively. When the concentration reached 10g / L, the remaining activity of the peroxidase was less than 20%. The inhibition degree of SDS is similar to that of CAB. At 1g / L, the relative activity of the enzyme remains at 60-80%. When the concentration reaches 10g / L, the relative enzyme activity remains at 20-40%. The non-ionic surfactants Tween-40, Tween-80, and Span-80 are different from the first two. At a concentration of 1g / L, the relative enzyme activities of the four peroxidases are all maintained above 95%. In particular, Span-80 has a promoting effect on peroxidase. The addition of 1g / L Span-80 increases the relative enzyme activities of RRLP, LCLP, PRLP and TFFP by 18.75%, 21.41%, 12.17% and 14.25%, respectively. Even when the concentration is as high as 10g / L, it can still maintain more than 90% of the relative enzyme activity. Therefore, it can be used as a stabilizer and promoter of peroxidase.

[0135] Physical and chemical properties

[0136] The ExPASyProtParam tool (https: / / www.expasy.org / resources / protparam) was used to calculate and predict the physicochemical properties of peroxidase, including molecular weight, total number of amino acids, molecular formula, atomic composition, theoretical isoelectric point (pI), total number of positively / negatively charged residues, lipid index, and overall average hydrophilicity.

[0137] Roystonearegia (RRLP, PDB: 3hdl) and Trachycarpusfortune were found in the PDB database.

[0138] The protein tertiary model of the peroxidase from RRLP (TFFP, PDB: 4usc) was used to predict and compare its physicochemical properties together with the PRLP model obtained by homology modeling using the ExPASyProtParam tool. The results are listed in Table 2. The total number of amino acids of RRLP, TFFP and PRLP is more than 300, the molecular weight is greater than 30kDa, and the theoretical isoelectric point is less than 7, indicating that these three proteins are acidic proteins, which is consistent with the above experimental results. The main amino acids of these three proteins are alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp) and cysteine ​​(Cys). The fat index is defined as the relative volume occupied by the aliphatic side chain in the protein. It is considered to be a positive factor in improving the thermal stability of the protein. The higher the value, the higher the stability of the enzyme. The fat index of RRLP, TFFP and PRLP is 88.39, 93.98 and 86.15, respectively. The results prove that these peroxidases are thermally stable within a certain temperature range. The overall average hydrophilicity is obtained by assigning positive values ​​to hydrophilic amino acids and negative values ​​to hydrophobic amino acids, adding the sum and dividing by the number of amino acids. The larger the value, the stronger the hydrophilicity. The hydrophilicity of TFFP is greater than that of RRLP. The overall average hydrophilicity of PRLP is negative, which means it is a hydrophobic protein.

[0139] Table 2 also lists the predicted molecular weights. Compared with the experimental data, the experimental molecular weights of RRLP, TFFP and PRLP are 39 kDa, 48 kDa and 50 kDa, respectively. The experimental molecular weights are larger than the theoretical molecular weights. The reason may be that different proteins have different abilities to bind SDS in SDS-PAGE electrophoresis, which will deviate from the normal value due to the inherent charge of the protein, and the protein will also migrate at different rates in the gel due to different solvent environments. Another possible explanation is that the protein is modified by glycosylation, which leads to a decrease in the ability to bind SDS and changes the protein Stokes radius. The migration rate is often greatly slowed down, so there is a certain deviation between the experimental molecular weight and the theoretical molecular weight. The experimental isoelectric points of RRLP, TFFP and PRLP are 5.1, 5.7 and 5.4, and the deviations from the theoretical isoelectric points are 0.04, 0.14 and -0.46, respectively. The model prediction is in good agreement with the experimental data.

[0140] Table 2 Prediction of the physicochemical properties of peroxidase

[0141]

[0142]

[0143] Application of peroxidase

[0144] Oxidation reaction of peroxidase and guaiacol

[0145] The specific operation steps of the oxidation reaction are as follows: add 0.1g guaiacol, 0.5mL H2O2 (0.1mol / L) and 0.01g PRLP to 10mL phosphate buffer solution (0.01mol / L, pH5) and react at room temperature for 2h. The product was determined by triple quadrupole gas chromatography-mass spectrometry (GC-MS, Agilent8890-7000D / Brukermaxisimpact, Agilent / Bruker, Germany), using a DP-5ms column and FID detector. The separation temperature was maintained at 40°C for the first 2min, then increased to 300°C at a rate of 10°C / min, and maintained at 300°C for 10min. After the addition of PRLP and H2O2, guaiacol was immediately oxidized and the color turned reddish brown. See Appendix Fig.10 It can be seen that the monomer (m / z = 124) polymerizes into dimers (m / z = 246) or trimers (m / z = 368), among which guaiacol dimer is the most abundant product, with four peaks at 21.2min, 23.2min, 23.4min and 23.6min with an m / z value of 246. The peaks at different positions are likely to correspond to phenolic or quinone intermediates (see Appendix Fig.11 ), and trimer appeared at 30.8min. Based on the above results, the inventors judged that the dimer was covalently linked through C5-C5 on the benzene ring of the monomer, and the trimer was connected by C3-C5 bond formed by free radical coupling between the dimer and the monomer. Based on this, it is speculated that the carbon-carbon bond connection of the benzene ring of the monomer is the main mechanism of PRLP / H2O2-catalyzed guaiacol polymerization.

[0146] Molecular docking of peroxidase and guaiacol

[0147] The use of computer programs to simulate the interaction between enzymes and ligands helps to deeply understand the principle of enzymatic reactions at the molecular level and helps to predict the upper limit of oxidative polymerization. The present invention uses AutoDockVina to perform molecular docking to obtain the binding energy and binding site of molecular docking between peroxidase and guaiacol. Fig.12 The binding energies of RRLP, TFFP and PRLP with guaiacol are -5.4 kcal / mol, -6.2 kcal / mol and -5.6 kcal / mol, respectively. The K of RRLP, TFFP and PRLP for guaiacol oxidation are m 3.63mmol·L -1 , 2.58mmol·L -1 and 3.13mmol·L -1 , the higher the absolute value of the binding energy with guaiacol, the higher the K m The smaller it is, the stronger its affinity for the substrate.

[0148] Through the exploration of this application, the applicant found that from the optimal conformation obtained by docking, it can be seen that guaiacol enters the catalytic channel of RRLP, hydrogen bonds with Arg38, Pro69 and Ser73 of RRLP, and binds with Pro139, Pro141, Ser178 and the heme group by hydrophobic force. The phenolic hydroxyl group of guaiacol simulated by docking is connected with the carboxyl group and Ser178 of the heme group of TFFP by hydrogen bonds, and binds with Ala68, Pro69, Pro141, Arg174 and Phe177 by hydrophobic force. The amino acid residues that hydrogen-bond guaiacol to PRLP are Arg70, Ser105, and Arg207. Other groups bound by hydrophobic forces include Glu100, Gln101, Ile170, Pro171, Pro173, and Asn210. In PRLP, there is a hydrophobic interaction between guaiacol and the heme cofactor. Guaiacol reacts with peroxidase to form free radicals, which collide to form dimers. The binding energy obtained after the dimer docks with the peroxidase is lower than that of the monomer. The dimer free radical collides with the monomer free radical to form a trimer. The trimer can also enter the catalytic channel of the peroxidase, and is more stable than the dimer and monomer. By simulating guaiacol polymers with higher polymerization degrees, the relationship between the degree of polymerization and the binding energy of the three peroxidases is obtained, see Appendix. Fig.13 The greater the degree of polymerization, the greater the absolute value of the binding energy. These enzymes can accommodate one end of the polymer into the catalytic center, thereby causing an oxidation reaction. When the degree of polymerization is 9 or 10 or more, the molecular size is larger and cannot enter the catalytic channel, so the absolute value of the binding energy decreases and the binding is weakened. In this experiment, no oligomers with a degree of polymerization > 4 were detected.

[0149] 3D model of peroxidase

[0150] Molecular dynamics simulation and modeling of the interaction between peroxidase and guaiacol

[0151] The molecular 3D structure of guaiacol was obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), saved in SDF format, opened in Chimera software and saved in PDB format.

[0152] Open the protein structure PDF file in Autodock software, perform dehydration, hydrogenation and charge balance processing, and export it as a PDBQT file. Export the ligand molecule as a PDBQT file in the Ligand option of Autodock software. Open the protein and ligand molecules in the Grid option and set the box size to The grid length is The box area can completely cover the protein. Set the docking parameters in the Docking option and run AutodockVina for molecular docking, with 20 dockings per set of conditions.

[0153] After the docking is completed, the best docking conformation is selected to generate a PDB format file, the results are redrawn and the size is measured using Chimera software, and the amino acid residues that interact with the ligand are analyzed using LigPlus software.

[0154] The translated amino acid sequence of Phoenix roebelenii was uploaded to the SWISS-MODEL server, and the template with the highest consistency and coverage was searched, with the PDB number of 4cuo (source: Ficus benghalensis, number of amino acids: 306, molecular weight: 37.7 kDa). The tertiary structure model of PRLP protein was established based on this template (see Appendix Fig.14 (b) in the figure). The model is generally consistent with the tertiary structure of the template, with only two irregular loop structures that differ ( Fig.14 (c) in the figure). The model is consistent with the catalytic center features of the template. The heme prosthetic group and the two calcium ions are bound at the same site, but the bound amino acid residues are slightly different. The amino acids that form hydrogen bonds with the heme prosthetic group in template 4cuo are Arg31, Ser35, His169, Arg173, Gln175 and Asp246, and the amino acids that interact with the two calcium ions are Asp43, Val46, Gly48, Asp50, Ser52 and Thr170, Asp221, Thr224, Ala227, Asp229. PRLP is hydrogen-bonded to the heme prosthetic group and residues of Arg63, His201, Val205, Arg207, and Asp278, and two calcium ions form hydrogen bonds with the amide or carboxyl groups of Asp75, Val78, Gly80, Asp82, Ser84 and Thr202, Asp253, Thr256, Leu259, and Asp261, respectively.

[0155] The SWISS-MODEL server scored the model quality as 45.70% identical to the template sequence of PRLP. Usually, when the consistency is greater than 40%, two proteins can be judged to be homologous proteins. The coverage rate is 90%, so it meets the requirements of protein homology modeling. The GMQE score is 0.75. This parameter is a score for the overall quality of the protein model. The score is between 0 and 1. The closer to 1, the more reliable the model. The QMEAN score is -1.28. This parameter is based on the local and overall quality estimation of the protein model. The absolute value between 0 and 2 can be judged as reliable model quality.

[0156] Table 3 Alignment of PRLP and 4cuo amino acid sequences

[0157]

[0158] Model Optimization

[0159] The reliability of the model structure (excluding auxiliary groups) was evaluated using the SAVES online server. The ERRAT module is a protein structure verification program determined by crystallography. The higher the overall quality score of ERRAT, the better the model quality. The ERRAT value of the PRLP model constructed using SWISS-MODEL in this study was 94.237, which is close to the high-resolution crystal structure (95%). On the 99% error line, Ile215, Tyr216, Asp218 and Leu221 are displayed in red and need to be optimized. The PROCHECK module uses RamachandranPlot analysis to evaluate the stereochemical quality of the protein structure residue by residue. The results show that 90.2% of the amino acids in the PRLP model are located in the optimal region, 8.6% are located in the additional allowed region, 1.1% of the amino acids are located in the loosely allowed region, and no amino acids are located in the unallowed region. The SAVES evaluation results show that the PRLP model constructed using the homology modeling method is more reasonable.

[0160] The three-dimensional structures constructed by homology modeling still have some quality problems and need to be optimized. The main purpose is to refine these low-resolution prediction models into high-resolution structures to reduce the deviation between the prediction model and the experimental structure. The 3DRefine online server program is based on the optimization of hydrogen bond networks and the force field to minimize the energy of the optimized model at the atomic level. The quality evaluation diagram of the optimized model is as follows: Fig.15 As shown in the figure, the optimized ERRAT score is 94.915, which eliminates the original Error red line in the ERRAT evaluation result, but adds Error red lines at Asp82 and Leu86. It can be seen that structural optimization cannot completely improve the quality of the model, which is closely related to the model structure itself and the prediction factors used by the optimization program. Fig.15 The RP diagram shows that 90.6% of the amino acids in the optimized model structure are located in the optimal region, 8.6% are located in the additional allowed region, 0.4% are located in the loosely allowed region, and 0.4% are located in the unallowed region.

[0161] The above are the specific experimental methods and preferred implementations of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A peroxidase, characterized in that: The peroxidase is extracted from natural plants and satisfies at least one of the following conditions (1) to (8): (1) The peroxidase activity ranged from 3.39 U / mL to 91.06 U / mL; (2) the protein content of peroxidase ranged from 0.405 to 0.736 mg / mL; (3) The specific activity of peroxidase ranged from 8.46 to 122.38 U / mg; (4) The isoelectric point of peroxidase is less than 7; (5) The relative molecular mass of peroxidase is between 39 kDa and 50 kDa; (6) The natural plant used to extract the peroxidase is at least one of royal palm leaves, fan palm leaves, soft-leafed thorny palm leaves, and palm fruits; and when the natural plants are royal palm leaves, palm fruits, and soft-leafed thorny palm leaves, the fat index of the peroxidase extracted from each of them is in the following order: palm fruits > royal palm leaves > soft-leafed thorny palm leaves; (7) With guaiacol as substrate, when the concentrations of FeCl3, MnCl2, ZnCl2, and FeSO4 were all 5 mmol / L, Mn 2+ , Fe 3+ 、Zn 2+ , Fe 2+ Ions have an inhibitory effect on the activity of peroxidase, and the order of inhibitory intensity is: Fe 2+ >Mn 2+ >Fe 3+ >Zn 2+ ; (8) 1 g / L Span-80 surfactant can increase the enzymatic activity of the peroxidase by at least 12%.

2. The peroxidase according to claim 1, characterized in that The total activities of peroxidase obtained from every 100 g of Livistona leaves, Eryngium leaves and palm fruits were 20.33 kU, 36.45 kU and 3.69 kU, respectively.

3. The peroxidase according to claim 1, characterized in that The optimum pH of peroxidase extracted from the leaves of Eryngium serrata is 4, while the optimum pH of peroxidase extracted from the leaves of royal palm, fan palm and palm fruit is 5.

4. The peroxidase according to claim 1, characterized in that When the system pH is 3-6, the relative enzyme activity of peroxidase extracted from the leaves of Livistona chinensis, Eryngium truncatum and palm fruit is above 60%.

5. The peroxidase according to claim 1, characterized in that The optimum catalytic temperature of the peroxidase is not less than 70°C.

6. The peroxidase according to claim 1, characterized in that The peroxidase can maintain more than 60% of the enzyme activity at a temperature of 90°C.

7. A method for extracting the peroxidase according to any one of claims 1 to 6, characterized in that: The steps include: A. Obtaining crude peroxidase solution The natural plant is collected to extract peroxidase, and the process includes: Wash with deionized water and drain; weigh 100g of leaves or fruits, cut the leaves to less than 2cm, mix with 150mL of phosphate buffer solution with a concentration of 0.01mol / L and pH 5, put into a plant wall breaking machine for high-speed wall breaking for 1min to obtain a homogenate, then filter with 8 layers of absorbent cotton gauze, and then centrifuge for 10min to remove the filter residue to obtain a clear liquid, which is used as the initial liquid for subsequent separation and extraction, recorded as water extract; slowly drip the water extract into a certain volume of acetone, stir while dripping, stand at 4℃ for 15min, centrifuge for 10min to remove the supernatant, obtain a precipitate, dissolve the precipitate with phosphate buffer, make the volume to 100mL, centrifuge again to remove insoluble matter, and obtain a crude enzyme solution; B. Purification of crude peroxidase solution The crude enzyme solution obtained by acetone extraction is pumped into an ion exchange chromatography column to remove pigments and impurities, specifically including: (1) Ion exchanger pretreatment: DEAE cellulose-DE52 was soaked in deionized water for 3 h, soaked in 0.5 mol / L NaOH solution for 30 min, then washed with deionized water until neutral, and then soaked in 0.5 mol / L HCl solution for 30 min and washed again until neutral; (2) Column loading: The pretreated DEAE cellulose-DE52 is loaded into the chromatography column, and the chromatography column is fully equilibrated with a 0.01 mol / L phosphate buffer solution with a pH of 5; (3) Sample loading: 7 mL of crude enzyme solution was added and balanced with 15 mL of phosphate buffer solution; (4) Elution and collection: During elution, 2 mol / L NaCl solution and phosphate buffer solution were used for gradient mixed elution. One tube of sample was collected every 5 minutes. The elution time was 2 hours, and a total of 24 tubes of samples were collected. The peroxidase activity and protein concentration of each tube of sample were detected. (5) Regeneration of the exchanger: Use 1 mol / L HCl and 1 mol / L NaOH for transformation regeneration. If the regenerated ion exchanger is not used for a long time, store it at 4°C to prevent microbial growth; C. Desalting and freeze drying of peroxidase The enzyme solution after ion exchange chromatography was desalted with an ultrafiltration centrifuge tube, and the enzyme solution was placed in an ultrafiltration centrifuge tube, centrifuged at 5000 rpm for 10 min, and the enzyme trapped in the centrifuge tube was dissolved with deionized water, centrifuged again, and the above operation was repeated several times until no Cl was detected in the centrifuge solution passing through the filter membrane. - Finally, the retained enzyme was dissolved in phosphate buffer and freeze-dried into powder using a vacuum freeze dryer to obtain peroxidase.

8. A method for extracting peroxidase according to claim 7, characterized in that: The extraction method also satisfies at least one of the following conditions: (1) In step A, the centrifugation condition is 4°C, 10000 rpm; (2) The diameter of the chromatography column in step B is 1.6 cm and the length is 30 cm.

9. Use of the peroxidase according to any one of claims 1 to 6 for catalysis or oxidation on guaiacol-containing materials.

10. The use according to claim 9, characterized in that The guaiacol-containing material is pulp for preparing paper.

Images