Peroxidase and application thereof

Abstract

The invention discloses peroxidase and application thereof, and relates to the field of bioengineering. The peroxidase POD-8 provided by the invention has very good enzyme activity for oxidizing phenol substrates, especially has the highest enzyme activity for 2, 6-DMP, has very good persistence, and can keep stable enzyme activity within 10 minutes; the resistance to various metal ions is relatively high, and the enzyme activity of 90% or above can still be kept in the presence of various metal ions with various concentrations; the POD-8 has a strong decolorizing capability on a plurality of dyes, especially has a decolorizing efficiency of about 25% on Congo red and amino black 10B in 24 h, and has a good application prospect in the aspect of dye wastewater treatment.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a peroxidase and an application thereof. Background Art

[0002] Phenolic compounds, a major pollutant in industrial wastewater (coking, pharmaceutical, and papermaking), are highly toxic and difficult to degrade. Traditional chemical oxidation methods carry the risk of secondary pollution, while enzymatic methods have become a research hotspot due to their mild conditions and high selectivity. However, natural peroxidases (such as horseradish peroxidase (HRP)) suffer from low catalytic efficiency and poor stability.

[0003] 1. Limitations of existing enzyme catalysis technology Insufficient activity: The conversion rate of natural HRP to phenolic substrates is only 40%-60%, and it is easily affected by pH and temperature; Cost bottleneck: Plant extraction requires large-scale cultivation, and recombinant expression yields are low (Saccharomyces cerevisiae expresses only 41 U / L of HRP). Limited application scenarios: The complex components of industrial wastewater (such as heavy metals and organic solvents) cause rapid enzyme inactivation. Although immobilization technologies such as gel encapsulation can improve stability, they will sacrifice more than 30% of activity.

[0004] 2. Technological breakthrough direction Recent research has focused on: Enzyme molecular modification: Improving the activity of the hemin prosthetic group through DNA-encoded microspheres (patent application publication number CN116251621A, which increased catalytic efficiency by 2.1 times), or rationally designing active center mutants (such as the patent application publication number CN119823976A, which disclosed the selective modification of the C-H bond oxidation of the AaeUPO enzyme); Nanozyme replacement: Tungsten nitride single-atom catalysts mimic enzyme active sites, achieving phenol degradation rates >90% and tolerating extreme pH; Cascade catalytic system: peroxidase and laccase are combined to synergistically oxidize lignin-derived phenolic polymers.

[0005] 3. Patent innovation space Current patents mostly focus on the immobilization or expression optimization of single enzymes, and lack systematic solutions for phenolic substrate specificity, environmental stress resistance, and industrial continuous production capabilities. Summary of the Invention

[0006] The invention provides a protein and nucleic acid molecule of a high-efficiency peroxidase. The enzyme can catalyze the oxidation of 2,6-DMP into 3,3',5,5'-tetramethyl-4,4'-diphenylquinone.

[0007] In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is as follows: First, the researchers discovered a candidate peroxidase protein and predicted its structure using AlphaFold. To verify its function and enzymatic properties, they fused the peroxidase to a His-SUMO tag via gene synthesis, recombinantly expressed it in Escherichia coli, and subjected it to Ni-NTA affinity chromatography to obtain the peroxidase candidate protein, POD-8.

[0008] The amino acid sequence of the peroxidase candidate protein is shown in SEQ ID NO.1.

[0009] On the other hand, the present invention also provides a nucleic acid molecule encoding the peroxidase.

[0010] Preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.2.

[0011] In yet another aspect, the present invention also provides a vector comprising the nucleic acid molecule.

[0012] Furthermore, in some embodiments of the present invention, the above-mentioned vector is a cloning vector or an expression vector; more preferably, it is a vector for use in bacteria, such as Escherichia coli ( E. coli ). Preferably, in some embodiments of the present invention, the expression vector is pRSFDuet1.

[0013] In yet another aspect, the present invention also provides a host cell comprising the vector.

[0014] The present invention also provides the use of the peroxidase, nucleic acid molecule, vector, or host cell in oxidizing substrates. The substrates include phenolic substrates such as 2,6-dimethylphenol or guaiacol, and non-phenolic substrates such as ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)).

[0015] The present invention explored the enzymatic activity of POD-8 on different substrates and tried three substrates: ABTS, guaiacol, and 2,6-dimethylphenol (2,6-DMP). The results showed that POD-8 had relatively good enzymatic activity on all three substrates. Among them, the enzymatic activity of 2,6-DMP as a substrate was higher than that of ABTS, and the enzyme activity stability was higher than that of guaiacol, maintaining stable enzyme activity within 10 minutes. Therefore, 2,6-DMP was selected as the substrate in subsequent experiments.

[0016] The present invention explored the optimum pH and temperature of POD-8 respectively and found that the optimum pH of POD-8 was 6.0, and the enzyme activity was maintained above 90% in the pH range of 6.0-7.0; the optimum temperature was 30°C, and the enzyme activity was maintained above 80% between 30°C and 50°C.

[0017] The present invention also provides a method for improving the decolorization efficiency of dyes, wherein the peroxidase is added to a reaction system containing metal ions, wherein the metal ions are Mn 2+ Mg 2+ 、Cu 2+ 、Na + , K + , Ca 2+ At least one of the above, with a final concentration between 0.5 and 10 mM.

[0018] The present invention explored the effects of different concentrations of different metal ions on the enzyme activity of POD-8. The results showed that 0.5 mM metal ions had a weak effect on the enzyme activity of POD-8, while higher concentrations of Mn 2+ 、Cu 2+ It has a significant promoting effect on POD-8, among which Mn 2+ and Cu 2+ The optimal concentrations of Zn were 1 mM and 5 mM respectively. 2+ 、Ni 2+ It has an inhibitory effect on POD-8, but 10 mM Ni 2+ The enzyme activity of POD-8 in the environment still remained above 85%, indicating that POD-8 has a strong tolerance to multiple metal ions.

[0019] The present invention also provides the use of the peroxidase, the nucleic acid molecule, the vector or the host cell in decolorizing synthetic dyes, wherein the synthetic dye is selected from at least one of Congo red, crystal violet, neutral red, amido black 10B, alizarin red and methyl orange.

[0020] Preferably, the application is implemented in a dye wastewater treatment system.

[0021] The present invention explored the decolorization effect of POD-8 on synthetic dyes and found that POD-8 has a decolorization effect on Congo red, neutral red, amido black 10B, crystal violet, alizarin red, and methyl orange. Among them, Congo red, amido black 10B, and crystal violet, which have the highest decolorization rates in 18 hours and 24 hours, all have decolorization rates of approximately 20%. The decolorization rates for neutral red, alizarin red, and methyl orange are relatively weak, at around 10%. In summary, POD-8 has a strong decolorization ability for a variety of dyes and has great application prospects in dye wastewater treatment.

[0022] Beneficial effects of the present invention: The peroxidase POD-8 provided by the present invention has excellent enzymatic activity for oxidizing phenolic substrates, especially the highest enzymatic activity for 2,6-DMP, and has very good sustainability; it has relatively strong tolerance to various metal ions and can still maintain more than 90% of the enzymatic activity in the presence of various metal ions; POD-8 has strong decolorization ability for various dyes, especially the decolorization efficiency of Congo red and amido black 10B is as high as about 25% within 24 hours, and has good application prospects in dye wastewater treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 In the present invention, AlphaFold is used to predict the three-dimensional structure of the peroxidase candidate protein.

[0024] Figure 2 This is a map of the peroxidase recombinant expression plasmid His-sumo-POD-pRSFDuet1.

[0025] Figure 3 The enzymatic activity of the peroxidase POD-8 of the present invention on different substrates.

[0026] Figure 4 This is a diagram showing the results of enzyme activity determination of peroxidase POD-8 at different pH values.

[0027] Figure 5 The diagram shows the results of enzyme activity determination of peroxidase POD-8 at different temperatures.

[0028] Figure 6 This figure shows the results of the determination of the effects of different metal ions on the activity of peroxidase POD-8.

[0029] Figure 7 This is a diagram showing the decolorization effect of peroxidase POD-8 on different dyes. DETAILED DESCRIPTION

[0030] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0031] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0032] The main experimental materials used in the following examples are from: Escherichia coli host strain E. coliBL21(DE3) was purchased from Beijing Qingke Biotechnology Co., Ltd., kanamycin was purchased from Dalian Baosheng Biotechnology Co., Ltd., and isopropyl-β-D-thiogalactopyranoside (IPTG) was a product of Promega. Protein purification and enzyme activity assay reagents were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0033] Example 1 Preparation of His-sumo-POD-pRSFDuet1.

[0034] 1. The present invention discovered a peroxidase candidate protein, the amino acid sequence of which is shown in SEQ ID NO.1, the nucleotide sequence of which is shown in SEQ ID NO.1, and predicted its protein structure using AlphaFold ( Figure 1 To verify its function and enzymatic properties, the peroxidase was fused to the downstream of the His-SUMO tag through gene synthesis, and GenScript Biotech Co., Ltd. was commissioned to synthesize the plasmid (such as Figure 2 The plasmid was transformed into BL21 (DE3) competent E. coli cells by heat shock method; the transformed cells were spread on LB plates containing 50 μg / mL kanamycin and cultured overnight, and positive clones were selected for further verification; 2. Expansion culture: Inoculate the selected positive clones into LB liquid medium containing antibiotics for expansion culture; culture at 37°C and 220 rpm until the logarithmic growth phase; 3. Induce expression: When the OD value of the bacterial solution reaches 0.6-0.8, add IPTG to the culture medium to a final concentration of 0.1mM and incubate at 20°C and 220 rpm for 20 hours to induce the expression of the target protein. 4. Centrifuge the induced bacterial solution, collect the E. coli precipitate, lyse the bacteria, release the intracellular proteins, and obtain the protein solution; 5. The lysed protein solution was loaded onto a pre-equilibrated Ni-NTA affinity chromatography column. Utilizing the specific binding of the His-Sumo tag to Ni ions, the column was gradient eluted with a buffer containing different concentrations of imidazole. The eluate containing the target protein was collected to preliminarily obtain a His-Sumo-tagged peroxidase, which was named POD-8.

[0035] Example 2 Substrate universality of POD-8.

[0036] POD-8 enzyme activity was determined using the 2,6-DMP, guaiacol, and ABTS assays. The change in absorbance over a 2-minute period was measured at 37°C. One unit (U) of enzyme activity was defined as the amount of enzyme required to convert 1 μmol of substrate per minute. Sodium malonate buffer, MnSO₄, substrate, H₂O₂, and enzyme were added to a 96-well plate. The reaction system is shown in Table 1.

[0037] Table 1

[0038] The results are as follows Figure 3 As shown in the figure, peroxidase POD-8 has good enzymatic activity against the three substrates, among which the enzymatic activity against 2,6-DMP is the highest, reaching 36.94 U / mg.

[0039] Example 3 Peroxidase enzyme activity assay.

[0040] Using 2,6-DMP as a substrate, an enzyme activity assay reaction system was prepared according to Example 2, and the absorbance change at 470 nm was measured at 37° C. within 10 minutes.

[0041] The calculation formula of peroxidase activity and absorbance change (ΔA) is: Activity (U / mg prot) = 672.04 × ΔA ÷ Cpr ÷ T; ΔA=A 2min -A 0min Cpr: enzyme concentration, obtained by measuring the absorbance of the peroxidase candidate protein at 280 nm and dividing it by its absorbance. T: reaction time, in minutes.

[0042] Table 2 Results of 2-min catalytic activity verification of purified peroxidase

[0043] Example 4 Determination of the optimal pH of peroxidase POD-8.

[0044] The optimal pH of peroxidase POD-8 was determined at 37°C using 2,6-DMP as substrate in a buffer system consisting of 50 mM tartaric acid-sodium tartrate buffer (pH 2.0-4.0), citric acid-sodium citrate buffer (pH 5.0-6.0), Tris-HCl buffer (pH 7.0-9.0), and glycine-NaOH (pH 10.0-11.0).

[0045] The results showed that the optimum pH for peroxidase POD-8 was 6.0, and the enzyme activity was maintained above 90% within the pH range of 6.0-7.0 ( Figure 4 ).

[0046] Example 5 Determination of the optimal temperature of peroxidase POD-8.

[0047] Using 2,6-DMP as substrate, purified peroxidase POD-8 was reacted in citric acid-sodium citrate buffer (pH 6.0) at temperatures of 20℃, 30℃, 40℃, 43℃, 45℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 2 min. The absorbance at 470nm was measured, and the enzyme activity was calculated to determine the optimal temperature of peroxidase POD-8.

[0048] The results showed that the optimum temperature for peroxidase POD-8 was 30℃ ( Figure 5 ), and maintain more than 80% enzyme activity between 30℃ and 50℃.

[0049] Example 6 Determination of the effects of metal ions on the activity of peroxidase POD-8.

[0050] Mn was added to each reaction mixture. 2+ Mg 2+ , Ca 2+ 、Cu 2+ 、Zn 2+ , K + 、Ni 2+ 、Na + 、Zn 2+ The final concentration of metal ions was 0.5–10 mM. The absorbance change of the reaction mixture at the optimal temperature and pH was measured over 10 minutes. Enzyme activity was calculated, with the peroxidase activity measured in the absence of metal ions (NC) as 100%. Three replicates were used for each group.

[0051] The results showed that 0.5 mM metal ions had a weak effect on the enzyme activity of POD-8, while higher concentrations of Mn 2+ 、Cu 2+ It has a significant promoting effect on POD-8, among which Mn 2+ and Cu 2+ The optimal concentrations of 1 mM and 5 mM were Figure 6 ). While higher concentrations of Zn 2 + 、Ni 2+ It has an inhibitory effect on POD-8, but 10 mM Ni 2+ The enzyme activity of POD-8 in the environment still remained above 85%, indicating that POD-8 has a strong tolerance to multiple metal ions.

[0052] Example 7 Decolorization of synthetic dyes by peroxidase POD-8.

[0053] A total of six dyes, including Congo red, neutral red, amido black 10B, crystal violet, alizarin red, and methyl orange, were prepared into 100 mg / L solutions using 50 mM citric acid-sodium citrate buffer (pH 6.0). The maximum absorption wavelength of the dyes was scanned in the range of 220-700 nm using an ultraviolet spectrophotometer.

[0054] Prepare the reaction system as shown in Table 3, with a final dye concentration of 100 mg / L. After 18 and 24 hours of reaction at the optimal temperature and pH, measure the absorbance of the reaction system using a UV spectrophotometer at the maximum absorbance wavelength of each dye. Set up a negative control group without enzyme addition.

[0055] Table 3

[0056] The dye decolorization rate is calculated using the following formula: R=(A0-A1) / A0×100%; R is the decolorization rate, A0 is the absorbance value of the control, and A1 is the absorbance value of the treatment group.

[0057] The results showed that peroxidase POD-8 had a decolorizing effect on Congo red, neutral red, amino black 10B, crystal violet, alizarin red and methyl orange ( Figure 7 Among them, Congo red, amino black 10B and crystal violet, which have the highest decolorization rates in 18 hours and 24 hours, have a decolorization rate of about 20%. The decolorization rates of neutral red, alizarin red and methyl orange are relatively weak, about 10%.

[0058] In conclusion, peroxidase POD-8 has a strong decolorization ability for a variety of dyes and has a good application prospect in dye wastewater treatment.

Claims

1. A peroxidase, characterized in that The amino acid sequence is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the peroxidase according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. A vector comprising the nucleic acid molecule according to claim 2 or 3. A host cell comprising the vector according to claim 4.

6. Use of the peroxidase according to claim 1, the nucleic acid molecule according to claim 2 or 3, the vector according to claim 4 or the host cell according to claim 5 in oxidizing a substrate; The substrate is 2,6-dimethylphenol, ABTS or guaiacol.

7. Use of the peroxidase according to claim 1, the nucleic acid molecule according to claim 2 or 3, the vector according to claim 4 or the host cell according to claim 5 in decolorizing a synthetic dye, wherein the synthetic dye is selected from at least one of Congo red, crystal violet, amido black 10B, neutral red, alizarin red, and methyl orange.

8. The use according to claim 7, characterized in that The application is implemented in a dye wastewater treatment system.

9. A method for improving dye decolorization efficiency, characterized in that: The peroxidase according to claim 1 is added to a reaction system containing metal ions, wherein the metal ions are Mn 2+ Mg 2+ 、Cu 2+ 、Na + , K + , Ca 2+ At least one of the following is used, and the final concentration is 0.5~10 mM.

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