Mining and application of a novel azoreductase SaCinD

Abstract

The application discloses mining and application of a novel azo reductase SaCinD and belongs to the field of environmental pollutant dye degradation. A novel azo reductase with azo dye decolorization function is screened from Staphylococcus aureus LZ-01, and the degradation capacity of the enzyme to dyes such as reactive black and acid red and the reduction activity of the enzyme to Cr(VI) are further characterized. The application also carries out site-directed mutagenesis on SaCinD, the mutant of which has enhanced reduction activity to various substrates and has a larger substrate utilization spectrum. The application first discloses a double-activation mechanism of copper(II) to azo reductase, including copper-induced gene expression up-regulation and enzyme activity enhancement, and provides a new strategy for the synergistic degradation and bioremediation of composite pollutants.

Description

Discovery and Application of a Novel Azo Reductase SaCinD Technical Field

[0001] This invention relates to the discovery and application of a novel azo reductase, SaCinD, which belongs to the field of environmental pollutant degradation. Background Technology

[0002] With the rapid development of industries such as textiles, printing and dyeing, and papermaking, azo dyes have become one of the main sources of water pollution. The global annual production of synthetic dyes reaches 700,000 tons, of which 10-15% is discharged into the environment along with heavy metals. Azo dyes contain an azo bond (-N=N-) structure, exhibiting high chemical stability and biodegradability. Their long-term presence in the environment poses a serious threat to ecosystems and human health. Traditional physicochemical treatment methods, such as activated carbon adsorption, chemical oxidation, and photocatalytic degradation, while effectively removing dyes, suffer from drawbacks such as high cost, secondary pollution, and high energy consumption. Microbial degradation, as an environmentally friendly and economical treatment method, has received widespread attention, with enzymes within the microorganisms being key factors in achieving degradation. However, currently reported azo reductases generally suffer from low catalytic efficiency and narrow substrate spectrum, making it difficult to meet practical application needs. Cu(II) is a common co-pollutant in the dye industry, and its toxicity to microorganisms is usually considered an obstacle to bioremediation; few studies have explored the positive role of Cu(II) in the microbial degradation of azo dyes. Therefore, developing efficient azoreductases suitable for complex environments is of great significance for the biological treatment of dye wastewater. Simultaneously, screening for azoreductases that can enhance enzyme activity in the presence of heavy metal Cu(II), and further improving the enzyme activity of azoreductases, will help solve the problem of complex pollution in practical applications. Summary of the Invention

[0003] This invention provides an azo reductase SaCinD mutant, based on the amino acid sequence shown in SEQ ID NO.1, by performing any of the following mutations:

[0004] (1) The tyrosine at position 24 was mutated to alanine to obtain mutant Y24A;

[0005] (2) The phenylalanine at position 98 was mutated to alanine to obtain mutant F98A;

[0006] (3) Mutate tryptophan at position 144 to alanine to obtain mutant W144A.

[0007] This invention provides a gene encoding the mutant.

[0008] The present invention provides an expression vector for expressing the mutant or carrying the gene.

[0009] The present invention provides recombinant microbial cells expressing the mutant.

[0010] In one embodiment, the microorganisms include, but are not limited to, Escherichia coli and Staphylococcus aureus.

[0011] In one embodiment, the recombinant microbial cells use Escherichia coli Rosetta (DE3) as the host and pET-28a(+) as the vector.

[0012] The present invention also provides a method for preparing the azo reductase SaCinD mutant.

[0013] In one embodiment, the method involves culturing the recombinant microbial cells in a culture medium for a period of time and collecting the azo reductase SaCinD mutant.

[0014] In one embodiment, the method involves culturing recombinant Escherichia coli expressing the azoreductase SaCinD mutant in LB medium until the OD600 value is ≥0.6, adding IPTG to a final concentration of 0.5 mM, and culturing overnight at 16°C for 16-20 hours.

[0015] In one embodiment, the culture is carried out at 37°C and 150 rpm.

[0016] The present invention also provides a method for degrading azo dyes.

[0017] In one embodiment, the method involves culturing the azo reductase SaCinD mutant or the lysate of the recombinant Escherichia coli at 37-45°C in an environment containing an azo dye.

[0018] In one embodiment, the reaction pH is 7.0-10.0.

[0019] In one embodiment, the enzyme concentration in the reaction system is ≥10 μM.

[0020] In one embodiment, the azo dye includes, but is not limited to, reactive black, acid red, or methyl orange.

[0021] In one implementation, the reaction lasts for at least 10 minutes, or at least 20 minutes, or at least 30 minutes, or at least 40 minutes.

[0022] In one embodiment, the environment containing the azo dye also contains 10 μM SaCinD, 1 mM NADH, 10 μM MMFMN, and 100 mM Tris-HCl buffer.

[0023] The present invention also provides a method for reducing Cr(VI).

[0024] In one embodiment, the method involves adding the azoreductase SaCinD mutant or the lysate of the recombinant Escherichia coli to a reaction system containing Cr(VI).

[0025] In one embodiment, the reaction system comprises 20 μM SaCinD, 300 μM NADH, 200 μM Cr(VI), and 50 mM Tris-HCl buffer.

[0026] The present invention also provides the application of the azo reductase SaCinD mutant or the lysate of the recombinant Escherichia coli in the degradation of azo dyes, the azo dyes including Reactive Black, Acid Red or Methyl Orange.

[0027] The present invention also provides the application of the azo reductase SaCinD mutant or the lysate of the recombinant Escherichia coli in the reduction of Cr(VI).

[0028] Beneficial effects:

[0029] (1) The present invention mutated the azo reductase SaCinD, which improved its catalytic degradation ability and substrate utilization range of azo dyes. The mutants F98A and Y24A increased the catalytic activity of acid red by 1.8 times and 1.6 times, respectively. F98A and Y24A also expanded the substrate utilization spectrum of SaCinD, and obtained 33% and 53% of methyl orange decolorization ability, respectively.

[0030] (2) This invention provides the application of azo reductase in the co-contamination of heavy metals and dyes, and reveals a novel Cu(II)-mediated activation effect, which solves the problem of co-contamination of heavy metals and azo dyes. Attached Figure Description

[0031] Figure 1 shows the enzyme activity assay of SaCinD in this invention; where A is the SDS-PAGE analysis of purified SaCinD protein; B and C are the flavin cofactor composition of SaCinD analyzed by HPLC; D is the actual decolorization diagram of Acid Red by SaCinD; E is the UV-Vis spectrum of Acid Red catalyzed by SaCinD; FH is the effect of temperature, pH and reaction time on the activity of SaCinD; I is the phylogenetic tree of representative nitroreductases, chromate reductases, azoreductases and SaCinD.

[0032] Figure 2 shows the different substrate selectivity of SaCinD for various substrates in this invention; where A is the reduction efficiency of SaCinD for different azo dyes; BC is the Michaelis-Menten kinetics of SaCinD for Reactive Black and Acid Red; DE is the degradation intermediates and pathways of Reactive Black analyzed by LC-MS; FG is the degradation intermediates and pathways of Acid Red analyzed by GC-MS; H is the reduction efficiency of SaCinD for Cr(VI); I is the Michaelis-Menten kinetics of SaCinD with Cr(VI); and J is the reduction pathway of Cr(VI).

[0033] Figure 3 shows the effect of Cu(II) on the activity of SaCinD in this invention; where AD represents the effect of Cu(II) on the reduction activity of SaCinD in black, acid red, methyl orange and Cr(VI); E represents the enzyme kinetics of SaCinD in the presence or absence of Cu(II); FG represents the fluorescence spectra of SaCinD with Cu(II) or Ni(II); and H represents the binding between SaCinD and Cu(II) determined by ITC.

[0034] Figure 4 shows the key active sites of SaCinD and the sites that bind to Cu(II) as revealed by the site-directed mutagenesis of this invention; where A is the structural superposition of SaCinD and the structural homology protein CinD; B is the secondary structure composition of SaCinD; C is the substrate channel prediction of SaCinD; D is the electrostatic potential surface comparison between the reported azo reductase YhdA and SaCinD; E and F are the decolorization efficiency of SaCinD mutants for acid red and methyl orange.

[0035] Figure 5 shows the circular dichroism spectrum of SaCinD and its mutants in this invention. Detailed Implementation

[0036] The present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. Any technical solution that can be conceived by those skilled in the art based on the present invention and in combination with common knowledge in the art shall fall within the scope of protection of the present invention.

[0037] The enzymes used in the following embodiments of the present invention are from the following sources:

[0038] Azoreductase SaCinD: The present invention discovered that nitroreductase with an amino acid sequence as shown in SEQ ID No.1 has the characteristics of azoreductase, therefore, the enzyme is named azoreductase SaCinD.

[0039] Detection method:

[0040] Azo reductase activity was determined based on the amount of azo dye substrate reduced by the enzyme. Specific conditions were as follows: 1 mL of reaction solution contained 10 μM of the enzyme SaCinD and 20 μM of the substrate azo dye (Acid Red, Reactive Black, or Methyl Orange). The reaction was carried out at 37°C, and samples were taken for testing after 40 minutes. The detection wavelengths for the azo dyes were 598 nm for Reactive Black, 510 nm for Acid Red, and 464 nm for Methyl Orange. The specific activity of azo reductase was defined as the number of micromoles of azo dye substrate degraded per milligram of enzyme per minute, expressed in μM / min / mg.

[0041] Assay for Cr(VI) reductase activity: The activity was determined based on the reduction of Cr(VI) substrate by the enzyme. The specific steps were as follows: The enzyme to be tested was reacted with Cr(VI) at 37°C, and a sample was taken at 10 minutes of reaction. The residual Cr(VI) concentration was measured at 540 nm using the diphenylcarbazide colorimetric method. The reaction system consisted of 20 μM SaCinD, 300 μM NADH, 200 μM Cr(VI), and 50 mM Tris-HCl buffer (pH 7.0). The specific activity of Cr(VI) reductase was defined as the number of micromoles of Cr(VI) that the enzyme could reduce per milligram per minute, expressed in μM / min / mg.

[0042] Determination of the effect of Cu(II) on the conformation of SaCinD: Different concentrations of metal ions were mixed with SaCinD and incubated at 37℃ for 5 min. The mixture was then transferred to a quartz cuvette to detect changes in fluorescence intensity. 1 mL of 20 mM Tris-HCl (pH 7.0) buffer contained 5 μM SaCinD, while the Cu(II) concentration was set to 0-100 μM and the Ni(II) concentration to 0-30 μM. The excitation wavelength was 280 nm, the scanning wavelength range was 300-450 nm, and the slit width was 5 nm. Synchronous fluorescence (Δλ, 15 nm or 60 nm) absorption spectroscopy was used to assess changes in protein microstructure.

[0043] Determination of binding kinetics between SaCinD and Cu(II): Isothermal titration calorimetry (ITC) was performed using a MicroCal PEAQ-ITC instrument. 20 μM SaCinD was placed in the sample cell, and 200 μM CuSO4 solution was added to the syringe. The titration protocol consisted of a single 0.4 μL injection, followed by 18 injections of 2 μL each, performed at a constant temperature of 25 °C. A 150-second delay was maintained between injections to ensure complete equilibration.

[0044] The decolorization rate is calculated by measuring the change in absorbance of the substance at the wavelength of maximum absorption. The calculation formula is: Decolorization rate = [(Original absorbance - Absorbance after treatment) / Original absorbance] * 100%.

[0045] Example 1: Heterologous expression, purification, and biochemical characterization of SaCinD protein

[0046] 1. Expression and purification of SaCinD protein

[0047] The SaCinD gene (sequence shown in SEQ ID No. 2) was cloned into the NcoI and XhoI restriction sites of the pET-28a expression vector, and the resulting recombinant plasmid pET-28a-SaCinD was transformed into *E. coli* Rosetta for expression. The resulting engineered bacteria were cultured in an atmosphere containing kanamycin (50 μg / mL). -1 ) and chloramphenicol (30 μg·mL -1 In LB medium, cultured at 37°C and 150 rpm until OD 600 The concentration of the protein was increased to 0.6, and IPTG was added to a final concentration of 0.5 mM. Expression was induced overnight at 16°C for 16-20 hours. After cell collection, the cells were resuspended in a buffer solution containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole, sonicated, and the supernatant was collected by centrifugation. The target protein was purified by Ni-NTA affinity chromatography according to standard procedures. SDS-PAGE electrophoresis showed that high-purity SaCinD protein with a molecular weight of approximately 24.8 kDa was obtained (Figure 1A). HPLC analysis confirmed that SaCinD contains flavin mononucleotide (FMN) as a cofactor (Figures 1B-C).

[0048] 2. Assay for SaCinD enzyme activity

[0049] To determine the azoreductase activity of SaCinD, an activity assay based on UV-Vis spectral changes was established. The standard reaction system contained 100 mM Tris-HCl, 10 μM SaCinD, 20 μM Acid Red, 10 μM FMN, and 1 mM NADH. Relative enzyme activities were measured under different reaction conditions.

[0050] Keeping the standard reaction system unchanged, with the buffer solution Tris-HCl pH value of 7.0 and the temperature of 37℃, the reaction time was changed (0-40 min), and the Ab value of the reaction solution was detected every 10 min. The results showed that SaCinD can efficiently catalyze the decolorization of Acid Red (Figure 1D-E). After 40 min of reaction, the Acid Red solution was obviously decolorized from light red to yellow (Figure 1D, H).

[0051] The above reaction was performed at different pH values ​​at a temperature of 37℃ and a reaction time of 40 min. The enzyme activity of azoreductase SaCinD was detected, with the highest detected enzyme activity taken as 100%, and the relative enzyme activity at other pH values ​​was calculated. The results showed that the optimal pH for azoreductase SaCinD was 7.0 (Figure 1G).

[0052] The above reaction was carried out at different temperatures under the conditions of Tris-HCl pH 7.0, reaction time 40 min, and keeping the standard reaction system unchanged. The enzyme activity of SaCinD azoreductase was detected, and the highest detected enzyme activity was taken as 100%. The relative enzyme activities at other temperatures were calculated. The results showed that the optimal reaction temperature for azoreductase SaCinD was 45℃ (Figure 1F).

[0053] Phylogenetic analysis was performed on SaCinD along with azo reductases and sequences of nitro reductases and chromium reductases, which have been reported to have the ability to reduce azo dyes. The phylogenetic tree was constructed using MEGA7 software via neighbor-joining and validated with 1000 replicates. The results showed that SaCinD clustered with the known azo reductase (PDB 1YWQ) in a single phylogenetic branch (Figure 1I).

[0054] 3. Substrate specificity of SaCinD

[0055] The substrate specificity of SaCinD was investigated by reacting the enzyme in a solution containing 100 mM Tris-HCl (pH 7.0), 10 μM SaCinD, 20 μM azo dye, 10 μM FMN, and 1 mM NADH at 37 °C for 40 min. The enzyme exhibited differentiated degradation efficiencies for different azo dyes, showing the highest degradation efficiency for Reactive Black and Acid Red, but lacking the ability to degrade methyl orange (Figure 2A). Enzyme kinetic analysis showed that SaCinD had an affinity of 30.13 μM for Acid Red and a maximum specific activity of 1.201 μM / min / mg, while its affinity for Reactive Black was 36.63 μM and its maximum specific activity was 10.968 μM / min / mg (Figures 2B-C).

[0056] The products and pathways of SaCinD degradation of azo dyes were analyzed using LC-MS and GC-MS. GC-MS analysis showed that the azo bond of Acid Red was broken, generating 5-imin-6-oxo-1,3-naphthalenedisulfonic acid (Fig. 2F, G). Similarly, LC-MS analysis of the degradation products of Reactive Black identified the products of azo bond cleavage as mono-[2-(4-aminobenzenesulfonyl)-ethyl] sulfate and 3,6-diamino-4-hydroxynaphthalene-2,7-disulfonate (Fig. 2D, E). Furthermore, enzyme kinetic analysis showed that SaCinD had an affinity for Cr(VI) of 163.4 μM and a maximum specific activity of 15.2 μM / min / mg, indicating that it also has the ability to reduce chromates (Fig. 2H-J).

[0057] 4. Effect of Cu(II) on SaCinD enzyme activity

[0058] Given that Cu(II) induces SaCinD expression in vivo, the effect of Cu(II) on its activity was further investigated. Enzyme activity was measured in 1 mL Tris-HCl buffer (100 mM, pH 7.0) containing 20 μM azo dye (Acid Red, Methyl Orange, or Reactive Black), 10 μM SaCinD, 1 mM NADH, 10 μM FMN, and 50 μM Cu(II) at 37 °C for 40 min. The results showed that Cu(II) significantly enhanced the catalytic activity of SaCinD, increasing the reduction efficiency of Acid Red and Cr(VI) by 1.4-fold and 2.4-fold, respectively, and broadened its substrate utilization spectrum to include the degradation of methyl Orange, increasing the reduction efficiency of methyl Orange from 4% to 38.4%, a 12.8-fold increase (Figures 3A-D). Enzyme kinetic analysis showed that Cu(II) increased the Kk of SaCinD for all tested substrates. m The decrease in the value indicates an enhanced substrate affinity for SaCinD. Simultaneously, the addition of Cu(II) increased the maximum specific activity of SaCinD for Acid Red from 1.201 μM / min / mg to 1.777 μM / min / mg (Figure 3E).

[0059] Example 2: Study on the mechanism of action of SaCinD and construction of mutants

[0060] 1. Interaction mechanism between Cu(II) and SaCinD

[0061] With increasing Cu(II) concentration, the fluorescence intensity of SaCinD significantly quenched, indicating a conformational change. Adding 7 μM Cu(II) reduced the fluorescence intensity to half its original value, indicating that SaCinD can bind Cu(II) (Figure 3F). Conversely, Ni(II) had no significant effect on the fluorescence intensity of SaCinD (Figure 3G). The binding constant of SaCinD to Cu(II) was 69.23 μM, accompanied by an enthalpy change (ΔH) of -2.8 × 10⁻⁶. 4 The cal·mol⁻¹ and entropy change (ΔS) are -76.4 cal·mol⁻¹·deg -1 (Figure 3H). This demonstrates that SaCinD specifically binds to Cu(II) and enhances its enzyme activity, thereby accelerating substrate reduction.

[0062] 2. Identification of key functional sites for SaCinD binding to Cu(II) and construction of mutants

[0063] To gain a deeper understanding of the substrate-binding pocket and its copper-binding site, the predicted monomer of SaCinD was analyzed using flavin-redoxin-like folding (Figure 4A-B). Structural analysis predicted the substrate channel of SaCinD, which possesses a hydrophobic substrate-binding pocket, facilitating the entry and binding of azo dyes (Figure 4C). Based on sequence alignment and protein channel analysis, nine key sites of SaCinD were identified. Using the recombinant plasmid pET-28a-SaCinD as a template, a linearized fragment containing a point mutation was obtained by PCR amplification using primers corresponding to F98A as shown in Table 1. The fragment was then circularized using the Blunting Kination Ligation Kit (Takara) to obtain plasmid pET-28a-SaCinD-F98A. Other point-mutated recombinant plasmids, pET-28a-SaCinD-Y24A, pET-28a-SaCinD-Q157A, pET-28a-SaCinD-K94A, pET-28a-SaCinD-Q140A, pET-28a-SaCinD-Y159A, pET-28a-SaCinD-W144A, pET-28a-SaCinD-L156A, and pET-28a-SaCinD-H158A, were obtained using the same method described above. The obtained mutant recombinant plasmids were transformed into Escherichia coli Rosetta and expressed and purified according to the method for wild-type SaCinD in Example 1.

[0064] Table 1 Primers required for constructing site-directed mutant strains of SaCinD protein.

[0065]

[0066]

[0067] Subsequently, following the method described in step 4 of Example 1, the decolorization of SaCinD wild-type and mutants with and without Cu(II) was tested for two representative pattern dyes, Acid Red and Methyl Orange. Regarding Acid Red decolorization, the Q140A, W144A, H158A, Q157A, and Y159A mutants showed a 10%–20% reduction in decolorization rate for Acid Red, indicating that these are conserved residues for the catalytic function of SaCinD (Figure 4E). The mutants F98A and Y24A increased the decolorization rate of SaCinD by approximately 30%. Notably, these mutants achieved methyl orange decolorization abilities of 33% and 53% activity, respectively (Figure 4F). Circular dichroism (CD) spectroscopy showed that F98A had the least impact on the overall structure of SaCinD, while Y24A significantly altered the structural integrity of SaCinD (Figure 5). The mutant Y24A exhibited high catalytic activity even without the addition of Cu(II), exceeding the activation effect of Cu(II) on the wild type, indicating its broader application potential in practical scenarios. Meanwhile, K94A and L156A maintained acid red reduction activity comparable to the wild type, but their activities decreased by 5% and 20%, respectively, upon the addition of Cu(II). This suggests that the K94 and L156 positions may play a crucial role in Cu(II) binding. These findings reveal key structure-function relationships and provide important information for designing next-generation azo reductases with higher catalytic efficiency and broader substrate specificity.

[0068] Table 2. Decolorization of dye by wild-type and mutant SaCinD

[0069]

[0070] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A SaCinD mutant of azo reductase, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.1, the following mutations are performed: tyrosine at position 24 is mutated to alanine; or phenylalanine at position 98 is mutated to alanine; or tryptophan at position 144 is mutated to alanine.

2. The gene encoding the mutant of claim 1.

3. Expressing the mutant of claim 1 or an expression vector carrying the gene of claim 2.

4. Recombinant microbial cells expressing the mutant of claim 1.

5. A recombinant Escherichia coli, characterized in that, The azo reductase SaCinD mutant of claim 1 was expressed using Escherichia coli Rosetta (DE3) as the host and pET-28a(+) as the vector.

6. The method for preparing the mutant of claim 1, characterized in that, The recombinant microbial cells of claim 4 or the recombinant Escherichia coli of claim 5 were cultured in a culture medium, and then the expression of the mutant was induced by IPTG.

7. A method for degrading azo dyes, characterized in that, The lysate of the mutant of claim 1 or the recombinant Escherichia coli of claim 5 is added to a reaction system containing an azo dye and reacted for at least 10 minutes, wherein the azo dye is acid red or methyl orange.

8. The method as described in claim 7, characterized in that, The reaction system also contains NADH, FMN and Tris-HCl buffer.

9. The application of the azo reductase SaCinD mutant of claim 1 or the lysate of the recombinant Escherichia coli of claim 5 in the degradation of azo dyes, characterized in that, The azo dye is either Acid Red or Methyl Orange.

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