A peroxidase and its use

Abstract

The application discloses a peroxidase and application thereof, and relates to the field of bioengineering.The peroxidase POD-8 provided by the application has good enzyme activity of oxidizing phenolic substrates, especially the highest enzyme activity on 2,6-DMP, and has very good persistence, can maintain stable enzyme activity within 10 min, and has relatively strong tolerance to various metal ions, can still maintain more than 90 % of enzyme activity in the presence of various metal ions with various concentrations, has relatively strong decolorizing capacity on various dyes, especially the decolorizing efficiency on Congo red and amino black 10B is as high as about 25 % within 24 h, and has a good application prospect in dye wastewater treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a peroxidase and its application. BACKGROUND

[0002] Phenolic compounds, as the main pollutants of industrial wastewater (coking, pharmaceutical, papermaking), have high toxicity and refractory characteristics. Traditional chemical oxidation method has the risk of secondary pollution, while biological enzyme method has become a research hotspot due to its mild conditions and high selectivity, but natural peroxidase (such as horseradish peroxidase HRP) has the problems of low catalytic efficiency and poor stability.

[0003] 1. Limitations of existing enzyme catalysis technology

[0004] Insufficient activity: the conversion rate of natural HRP to phenolic substrates is only 40%-60%, and it is easily affected by pH and temperature;

[0005] Cost bottleneck: large-area planting is required for enzyme production by plant extraction method, and the yield of recombinant expression is low (only 41 U / L of HRP expressed by Saccharomyces cerevisiae);

[0006] Limited application scenarios: complex components (such as heavy metals and organic solvents) in industrial wastewater cause rapid inactivation of enzymes, and although immobilization technologies such as gel embedding can improve stability, more than 30% of activity will be sacrificed.

[0007] 2. Breakthrough direction of technology

[0008] In recent years, research has focused on:

[0009] Enzyme molecule modification: improving the activity of chlorin heme cofactor through DNA encoding microspheres (patent application with publication number CN116251621A, catalytic efficiency improved by 2.1 times), or rationally designing active center mutants (such as patent application with publication number CN119823976A, which discloses the selective modification of C-H bond oxidation of AaeUPO enzyme);

[0010] Nanoenzyme replacement: tungsten nitride monatomic catalyst simulates the active site of enzyme, achieving phenolic degradation rate >90% and tolerance to extreme pH;

[0011] Cascade catalytic system: peroxidase and laccase are used together to synergistically oxidize lignin-derived phenolic polymers.

[0012] 3. Patent innovation space

[0013] Current patents mainly focus on single enzyme immobilization or expression optimization, and lack of systematic solutions for phenolic substrate specificity, environmental stress tolerance, and industrial continuous production capacity. SUMMARY

[0014] The present application provides a kind of high-efficiency peroxidase protein and nucleic acid molecule, which can catalyze 2,6-DMP oxidation into 3,3',5,5'-tetramethyl-4,4'-benzoquinone.

[0015] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0016] Firstly, the present application finds a peroxidase candidate protein, and predicts its protein structure using AlphaFold. To verify its function and enzymatic properties, the peroxidase is fused downstream of the His-sumo tag by gene synthesis, expressed in Escherichia coli, and subjected to affinity chromatography using Ni-NTA to obtain the peroxidase candidate protein POD-8.

[0017] The amino acid sequence of the peroxidase candidate protein is shown in SEQ ID NO. 1.

[0018] On the other hand, the present application also provides a nucleic acid molecule encoding the peroxidase.

[0019] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 2.

[0020] In another aspect, the present application also provides a vector comprising the nucleic acid molecule.

[0021] Further, in some embodiments of the present application, the above-mentioned vector is a cloning vector or an expression vector; more preferably a recombinant expression vector for expression in bacteria, such as Escherichia coli (E. coli). E. coli Preferably, in some embodiments of the present application, the expression vector is pRSFDuet1.

[0022] In another aspect, the present application also provides a host cell comprising the vector.

[0023] The present application also provides the use of the peroxidase, the nucleic acid molecule, the vector or the host cell in oxidizing a substrate. The substrate includes 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)).

[0024] The present application explores the enzyme activity of POD-8 on different substrates, and attempts three substrates: ABTS, guaiacol and 2,6-dimethylphenol (2,6-DMP). The results show that POD-8 has good enzyme activity on all three substrates, and the enzyme activity on 2,6-DMP as a substrate is higher than that on ABTS, and the enzyme activity stability is higher than that on guaiacol, maintaining stable enzyme activity within 10 min. Therefore, 2,6-DMP is selected as the substrate in subsequent experiments.

[0025] The application respectively explores the optimum pH and temperature of POD-8, and finds that the optimum pH of POD-8 is 6.0, the enzyme activity is maintained at more than 90% in the pH range of 6.0-7.0; the optimum temperature is 30 DEG C, and the enzyme activity is maintained at more than 80% between 30 DEG C and 50 DEG C.

[0026] The application also provides a method for improving the decolorization efficiency of dyes, wherein the peroxidase is added in a reaction system containing metal ions, the metal ions are at least one of Mn 2+ , Mg 2+ , Cu 2+ , Na + , K + , Ca 2+ , and the final concentration is between 0.5-10 mM.

[0027] The application explores the influence of different concentrations and different metal ions on the enzyme activity of POD-8, and the results show that 0.5 mM metal ions have weak influence on the enzyme activity of POD-8, higher concentrations of Mn 2+ , Cu 2+ have obvious promoting effect on POD-8, wherein the optimum concentrations of Mn 2+ and Cu 2+ are 1 mM and 5 mM respectively, and higher concentrations of Zn 2+ , Ni 2+ have inhibitory effect on POD-8, but the enzyme activity of POD-8 in the environment of 10 mM Ni 2+ still maintains more than 85%, which indicates that POD-8 has strong tolerance to various metal ions.

[0028] The application also provides the application of the peroxidase, the nucleic acid molecule, the vector or the host cell in the synthesis of dye decolorization, and the synthetic dyes are at least one of Congo red, crystal violet, neutral red, amino black 10B, alizarin red and methyl orange.

[0029] Preferably, the application is implemented in a dye wastewater treatment system.

[0030] The application explores the decolorization effect of POD-8 on synthetic dyes, and finds that POD-8 has decolorization effect on Congo red, neutral red, amino black 10B, crystal violet, alizarin red and methyl orange, wherein the decolorization rates of Congo red, amino black 10B and crystal violet which have the highest decolorization rates in 18 hours and 24 hours are about 20%, the decolorization rates on neutral red, alizarin red and methyl orange are weak, about 10%, in general, POD-8 has strong decolorization capacity on various dyes, and has good application prospect in dye wastewater treatment.

[0031] The application has the following beneficial effects:

[0032] The peroxidase POD-8 provided by the application has good enzyme activity of oxidizing phenolic substrates, especially the highest enzyme activity of 2,6-DMP, and very good persistence; the POD-8 has relatively strong tolerance to various metal ions, and can still maintain more than 90% of the enzyme activity in the presence of various metal ions; the POD-8 has relatively strong decolorization ability to various dyes, especially the decolorization efficiency of 25% or so of Congo red and amino black 10B in 24 hours, and has a good application prospect in dye wastewater treatment. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The application utilizes AlphaFold to predict the three-dimensional structure of peroxidase candidate proteins.

[0034] Figure 2 The figure of the peroxidase recombinant expression plasmid His-sumo-POD-pRSFDuet1.

[0035] Figure 3 The application provides the enzyme activity effect of the peroxidase POD-8 on different substrates.

[0036] Figure 4 The application provides the enzyme activity determination result graph of the peroxidase POD-8 under different pH values.

[0037] Figure 5 The application provides the enzyme activity determination result graph of the peroxidase POD-8 under different temperatures.

[0038] Figure 6 The application provides the determination result graph of the influence of different metal ions on the activity of the peroxidase POD-8.

[0039] Figure 7 The application provides the decolorization effect graph of the peroxidase POD-8 on different dyes. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the embodiments of the application clearer, the technical scheme in the embodiments of the application will be described clearly and completely below. If the specific conditions are not indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturer are adopted. If the reagents or instruments are not indicated by the manufacturer, they are all conventional products that can be purchased in the market.

[0041] The features and performances of the application will be further described in detail below in combination with the embodiments.

[0042] The main experimental materials used in the following embodiments are from:

[0043] Escherichia coli host strain E. coliBL21(DE3) was purchased from Beijing Genki Biological Technology Co., Ltd., kanamycin was purchased from Dalian Baosheng Biological Co., Ltd.; isopropyl-β-D-thiogalactopyranoside (IPTG) was a product of Promega Corporation. The reagents for protein purification and enzyme activity determination were purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0044] Example 1

[0045] Preparation of His-sumo-POD-pRSFDuet1.

[0046] 1. The present application finds a peroxidase candidate protein, the amino acid sequence is shown as SEQ ID NO. 1, the nucleotide sequence is shown as SEQ ID NO. 1, and the protein structure thereof is predicted by AlphaFold (https: / / alphafold.org / ). Figure 1 To verify its function and enzymatic properties, the peroxidase was fused downstream of the His-sumo tag by gene synthesis, and the plasmid was synthesized by Jinshuibiotech Co., Ltd. (as shown in Figure 2 The plasmid was transformed into BL21 (DE3) E. coli competent cells by heat shock method; the transformed cells were plated on LB plates containing 50 μg / mL kanamycin and cultured overnight, and positive clones were selected for further verification;

[0047] 2. Scale-up culture: the selected positive clones were inoculated into LB liquid medium containing antibiotics for scale-up culture; incubate at 37℃ 220 rpm until the logarithmic growth phase;

[0048] 3. Induction 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 0.1 mM, 20℃ 220 rpm for 20h to induce the expression of target protein;

[0049] 4. Centrifuge the induced bacterial solution, collect the E. coli precipitate, lyse the bacteria, release the intracellular protein, and obtain the protein solution;

[0050] 5. Load the lysed protein solution onto a pre-equilibrated Ni-NTA affinity chromatography column, use the specific binding of His-Sumo tag and Ni ion, elute with buffer containing different concentrations of imidazole, collect the eluate containing target protein, and preliminarily obtain the peroxidase with His-sumo tag, and the peroxidase is named as POD-8.

[0051] Example 2

[0052] Substrate universality of POD-8.

[0053] 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.

[0054] Table 1

[0055]

[0056] 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.

[0057] Example 3

[0058] Peroxidase enzyme activity assay.

[0059] 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.

[0060] The calculation formula of peroxidase activity and absorbance change (ΔA) is:

[0061] Activity (U / mg prot) = 672.04 × ΔA ÷ Cpr ÷ T;

[0062] 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.

[0063] Table 2 Results of 2-min catalytic activity verification of purified peroxidase

[0064]

[0065] Example 4

[0066] Determination of the optimal pH of peroxidase POD-8.

[0067] 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).

[0068] The results show that the optimum pH of peroxidase POD-8 is 6.0, and the enzyme activity is maintained at more than 90% in the pH range of 6.0-7.0. Figure 4 ).

[0069] Example 5

[0070] Determination of the optimum temperature of peroxidase POD-8.

[0071] Using 2,6-DMP as the substrate, in the citric acid-sodium citrate buffer at pH 6.0, the purified peroxidase POD-8 was incubated at temperatures of 20°C, 30°C, 40°C, 43°C, 45°C, 50°C, 60°C, 70°C, 80°C, and 90°C, respectively, and the absorbance at 470 nm was measured after 2 min of reaction. The enzyme activity was calculated to determine the optimum temperature of peroxidase POD-8.

[0072] The results show that the optimum temperature of peroxidase POD-8 is 30°C ( Figure 5 ), and the enzyme activity is maintained at more than 80% between 30°C and 50°C.

[0073] Example 6

[0074] Determination of the effect of metal ions on the activity of peroxidase POD-8.

[0075] In each reaction mixture, Mn 2+ , Mg 2+ , Ca 2+ , Cu 2+ , Zn 2+ , K + , Ni 2+ , Na + , and Zn 2+ metal ions were added, with a final concentration of 0.5-10 mM. The change in absorbance of the reaction mixture was measured within 10 min at the optimum temperature and pH, and the enzyme activity was calculated, with the enzyme activity of peroxidase determined without metal ions (NC) as 100%. Each group had three parallel samples.

[0076] The results show that 0.5 mM metal ions have a weak effect on the enzyme activity of POD-8, and higher concentrations of Mn 2+ , Cu 2+ have a significant promoting effect on POD-8, with the optimum concentrations of Mn 2+ and Cu 2+ being 1 mM and 5 mM, respectively ( Figure 6 ). Higher concentrations of Zn 2 + , Ni 2+POD-8 was inhibited, but 10 mM Ni 2+ The enzyme activity of POD-8 in the environment still maintained more than 85%, indicating that POD-8 had relatively strong tolerance to various metal ions.

[0077] Example 7

[0078] Decolorization of synthetic dyes by peroxidase POD-8.

[0079] Six dyes, Congo red, neutral red, amino black 10B, crystal violet, alizarin red, and methyl orange, were prepared into 100 mg / L solutions with 50 mM pH 6.0 citric acid-sodium citrate buffer. The maximum absorption wavelength of the dyes was scanned in the range of 220-700 nm using a UV spectrophotometer.

[0080] The reaction system was prepared as shown in Table 3, and the final concentration of the dye was 100 mg / L. After 18 h and 24 h of reaction at the optimum temperature and pH, the absorbance of the reaction system was detected at the maximum absorption wavelength of the different dyes using a UV spectrophotometer, and a negative control group without enzyme was set.

[0081] Table 3

[0082]

[0083] The decolorization rate of the dye was calculated using the following formula:

[0084] R = (A0-A1) / A0 x 100%;

[0085] R is the decolorization rate, A0 is the absorbance of the control, and A1 is the absorbance of the treatment group.

[0086] The results showed that peroxidase POD-8 had decolorization effects on Congo red, neutral red, amino black 10B, crystal violet, alizarin red, and methyl orange. Figure 7 Among them, the highest decolorization rates of Congo red, amino black 10B, and crystal violet were about 20% after 18 h and 24 h, and the decolorization rates of neutral red, alizarin red, and methyl orange were weaker, about 10%.

[0087] In summary, peroxidase POD-8 has strong decolorization ability for various dyes and has good application prospects 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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