A method for decolorizing dyes using peroxidase

Abstract

This invention discloses a method for degrading dyes using dye decolorization peroxidase, belonging to the field of biotechnology. This invention expresses a DyP (RhDyPB) derived from the bacterium *Rhodococcus jostii* in *Escherichia coli*. By adjusting the pH, manganese ion, hydrogen peroxide, and Tween-80 content during the decolorization process, the decolorization efficiency of RhDyPB and *E. coli* expressing RhDyPB on the anthraquinone dye Reactive Blue 19, the oxanthracene dye Eosin Y, the indigo dye Indigo Carmine, and the triphenylmethane dye Malachite Green is effectively improved.

Description

Technical Field

[0001] This invention relates to a method for degrading dyes using dye decolorization peroxidase, belonging to the field of biotechnology. Background Technology

[0002] Dyes are widely used in industries such as papermaking, textiles, and food. Based on their chemical structure, they can be classified into anthraquinone, azo, indigo, triphenylmethane, xanthracene, and nitro dyes, among others. Approximately 10-15% of dyes are discharged into the environment annually during production and use, often through wastewater. Chemically synthesized dyes are typically toxic, stable, and difficult to degrade naturally. Untreated dyes accumulate in the environment, ultimately damaging aquatic ecosystems and endangering human health. Therefore, it is essential to develop effective methods to address the problems caused by industrial waste dyes.

[0003] Currently, methods for treating dye wastewater include physical, chemical, and biological methods. Physical and chemical methods, such as membrane separation, adsorption, and ozone oxidation, have disadvantages such as high cost, production of concentrated sludge and byproducts, and environmental impact. Biological methods, on the other hand, are low-cost and do not cause additional pollution. Enzyme treatment is an effective biological method for dye decolorization, using one or more enzymes to break down the chromophores of the dye into smaller, less toxic molecules.

[0004] Dye decolorizing peroxidases (DyPs) are novel peroxidases that use heme as a cofactor, named for their ability to decolorize dyes. Unlike other peroxidase superfamily members, the amino acid sequence of DyPs does not share homology with other classic heme peroxidases. Although the first DyP was discovered in fungi, subsequent studies of bacterial DyPs have facilitated the heterologous expression and structural properties of DyPs. The literature "Characterization and use of abacterial lignin peroxidase with an improved manganese-oxidative activity; DOI:10.1007 / s00253-018-9409-3" discloses that RhDyPB has a good decolorizing effect, but its decolorizing effect on azo and methylene blue dyes is poor, with a decolorization rate of only 11% for methylene blue dyes. Compared with theoretical research, the influence of environmental factors on DyP decolorization in practical applications is still unclear. Meanwhile, the literature "Converting cytochrome cinto a DyP-like metalloenzyme; DOI:10.1039 / d2dt02137d" indicates that decolorization using recombinant bacteria expressing DyP has not yet been successful.

[0005] Therefore, there is an urgent need to find a method for efficient dye decolorization, to provide ideas for biological dye decolorization, and to help solve the serious problem of pigment pollution in the industrial and environmental fields. Summary of the Invention

[0006] To address the aforementioned problems, this invention expresses a DyP (RhDyPB) derived from the bacterium Rhodococcus jostii in Escherichia coli. By adjusting the manganese ion concentration, hydrogen peroxide concentration, pH value, and Tween concentration during the decolorization process, the decolorization efficiency of RhDyPB and the RhDyPB-expressing Escherichia coli on anthraquinone dye Reactive Blue 19, oxanthracene dye Eosin Y, indigo dye Indigo Carmine, and triphenylmethane dye Malachite Green is effectively improved.

[0007] The first objective of this invention is to provide a method for degrading dyes using dye decolorizing peroxidase (RhDyPB), comprising the steps of: adding dye decolorizing peroxidase to a decolorization reaction system, the decolorization reaction system comprising 2-6 mM NCl2 and 0.1-2 mM H2O2; the pH of the decolorization system being 4.5-6.0.

[0008] In one embodiment, the amino acid sequence of RhDyPB is identical to that of the amino acid accession number AYJ72200.1.

[0009] In one implementation, RhDyPB is obtained through heterologous expression in Escherichia coli.

[0010] In one embodiment, the preparation method of RhDyPB is as follows: the RhDyPB gene is integrated into the NdeI and XhoI sites of the pET-24b plasmid, and the glutamyl-tRNA reductase hemA, which encodes a key enzyme in the synthesis of heme in Escherichia coli, is integrated into the XbaI site. The hemA gene is located before the RhDyPB gene, thus constructing the plasmid pE24AD; the plasmid pE24AD is transformed into Escherichia coli JM109 for amplification, and then transformed into Escherichia coli BL21(DE3) to construct the recombinant strain Eco / pE24AD; RhDyPB is obtained through induced expression, isolation and purification.

[0011] In one embodiment, the amino acid sequence of hemA is identical to that of accession number CAQ31712.1.

[0012] In one embodiment, the decolorization reaction system uses sodium acetate or sodium malonate at a concentration of 45–50 mM as a buffer solution.

[0013] In one embodiment, the concentration of dye decolorizing peroxidase in the decolorization reaction system is 0.8–1.8 U / mL.

[0014] In one embodiment, the dye concentration in the decolorization reaction system is 20–60 mg / L.

[0015] In one embodiment, the decolorization reaction is carried out at 20–25°C for 10–24 hours.

[0016] In one embodiment, the dye is anthraquinone dye Reactive Blue 19, oxane dye Eosin Y, indigo dye Indigo Carmine, and triphenylmethane dye Malachite Green.

[0017] A second objective of this invention is to provide a method for whole-cell decolorization, comprising the steps of: adding bacterial cells expressing dye decolorization peroxidase to a decolorization reaction system, the decolorization reaction system comprising 2-6 mM MnCl2, 0.1-2 mM H2O2, and 0.5-5% (v / v) Tween-80; the pH of the decolorization system being 4.5-6.0.

[0018] In one embodiment, the dye is anthraquinone dye Reactive Blue 19, oxane dye Eosin Y, indigo dye Indigo Carmine, and triphenylmethane dye Malachite Green.

[0019] In one embodiment, the decolorization reaction system uses sodium acetate or sodium malonate at a concentration of 45–50 mM as a buffer solution.

[0020] In one embodiment, the enzyme content of the bacteria in the decolorization system is 2.2–3.0 U / mL.

[0021] In one embodiment, the dye concentration in the decolorization reaction system is 20–60 mg / L.

[0022] In one embodiment, the decolorization reaction is carried out at 20–25°C for 10–24 hours.

[0023] A third objective of this invention is to provide a method for degrading dyes. When the dye is anthraquinone dye Reactive Blue 19 or triphenylmethane dye Malachite Green, dye decolorization peroxidase is used for decolorization. The decolorization reaction system includes 2–6 mM MnCl2 and 0.1–2 mM H2O2, and the pH of the decolorization system is 4.5–6.0. ​​When the dye is oxane dye Eosin Y or indigo dye Indigo Carmine, cells expressing dye decolorization peroxidase are used for decolorization. The decolorization reaction system includes 2–6 mM MnCl2, 0.1–2 mM H2O2, and 0.5–5% (v / v) Tween-80, and the pH of the decolorization system is 4.5–6.0.

[0024] The fourth objective of this invention is to provide a method for improving the dye decolorization effect of dye decolorization peroxidase, comprising the steps of: adding dye decolorization peroxidase to a decolorization reaction system, wherein the decolorization reaction system comprises 2-6 mM NCl2 and 0.1-2 mM H2O2; and the pH of the decolorization system is 4.5-6.0.

[0025] The present invention also provides a method for improving the decolorization effect of whole cells, characterized by comprising the steps of: adding bacterial cells expressing dye decolorization peroxidase to a decolorization reaction system, the decolorization reaction system comprising 2-6 mM MnCl2, 0.1-2 mM H2O2, and 0.5-5% (v / v) Tween-80; the pH of the decolorization system being 4.5-6.0.

[0026] In one embodiment, the dyes are anthraquinone dye Reactive Blue 19, oxane dye Eosin Y, indigo dye Indigo Carmine, and triphenylmethane dye Malachite Green.

[0027] Beneficial effects

[0028] This invention expresses a DyP (RhDyPB) derived from the bacterium Rhodococcus jostii in Escherichia coli. By adjusting the pH, H2O2, manganese ion, and Tween-80 content during the decolorization process, the decolorization efficiency of RhDyPB and Escherichia coli expressing RhDyPB on anthraquinone dye Reactive Blue 19, oxanthracene dye Eosin Y, indigo dye Indigo Carmine, and triphenylmethane dye Malachite Green is effectively improved.

[0029] Specifically, when the amount of manganese ions added was 6 mM and the amount of H2O2 added was 2 mM, the decolorization rates of the anthraquinone dye Reactive Blue 19 and the triphenylmethane dye Malachite using RhDyPB reached 62.3% and 94.4%, respectively. When the amount of manganese ions added was 6 mM, the amount of H2O2 added was 2 mM, and the amount of Tween-80 added was 5% (v / v), the whole cells of recombinant Escherichia coli expressing RhDyPB showed better decolorization effect than enzyme decolorization on the oxanthracene dye Eosin Y and the indigo dye Indigo Carmine. When the pH was 6.0, the decolorization rates reached 95% and 92.4%, respectively. Attached Figure Description

[0030] Figure 1 The results of decolorization of different dyes in Comparative Example 1 are shown. Among them, a, b, c, and d are the decolorization results of anthraquinone dye Reactive Blue 19, oxanthracene dye Eosin Y, indigo dye Indigo Carmine, and triphenylmethane dye Malachite Green, respectively. The left side of each group is after decolorization, and the right side is before decolorization. Detailed Implementation

[0031] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0032] 1. Enzyme activity detection method:

[0033] Using 2,2'-azido-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (ε420nm=36000M) -1 cm -1 The activity of RhDyPB was measured at pH 4.5 as a substrate. One unit of enzyme activity (U) is defined as the amount of enzyme required to oxidize 1 μmol ABTS per minute at 25°C.

[0034] 2. Decolorization rate test method:

[0035]

[0036] Where A0 is the absorbance of the solution containing no enzymes or cells, A i The absorbance is the value of the supernatant after RhDyPB or cell treatment.

[0037] Example 1: Decolorization by peroxidase RhDyPB

[0038] A method for decolorization using peroxidase RhDyPB includes the following steps:

[0039] (1) Construction of recombinant bacteria for dye decolorization peroxidase (RhDyPB)

[0040] Using *E. coli* BL21(DE3) as the expression host, the full-length nucleic acid sequence of RhDyPB was chemically synthesized (the amino acid sequence is consistent with the accession number AYJ72200.1, and the codons were optimized), and inserted between the restriction sites NdeI and XhoI of plasmid pET-24b. To increase the endogenous level of the cofactor heme, the key enzyme encoding heme synthesis in *E. coli*, gamma-glutamyl-tRNA reductase hemA (the amino acid sequence is consistent with the accession number CAQ31712.1), was also integrated into the XbaI site, located before the RhDyPB gene, to construct plasmid pE24AD. pE24AD was transformed into *E. coli* JM109 for amplification, and then transformed into *E. coli* BL21(DE3) to construct the recombinant strain Eco / pE24AD.

[0041] (2) Expression and purification of RhDyPB

[0042] The Eco / pE24AD constructed in step (1) was inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 rpm for 12 h. After culture, it was inoculated into TB medium containing 50 μg / mL kanamycin at a 4% (v / v) inoculation rate and cultured at 37°C until OD200. 600 At a concentration of 1.0, 0.25 mM IPTG was added for induction, and the cells were further cultured at 20 °C for 18 h. The cells were harvested by centrifugation, washed, and resuspended in 50 mM phosphate buffer (pH 7.4) for ultrasonic disruption. The disrupted solution was centrifuged at 10950 g for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter to obtain the crude enzyme solution. The crude enzyme solution was then loaded onto a Ni-NTA column pre-equilibrated with phosphate buffer (pH 7.4, 20 mM sodium phosphate, containing 500 mM NaCl and 20 mM imidazole). A 20-500 mM linear imidazole gradient was used for elution to obtain purified RhDyPB, and the protein concentration was determined using the Bradford method. The purified RhDyPB concentration was 3.26 mg / mL, and the enzyme activity was 53.7 U / mL.

[0043] (3) The purified RhDyPB from step (2) was used to decolorize the anthraquinone dye Reactive Blue 19, the oxanthracene dye Eosin Y, the indigo dye Indigo Carmine, and the triphenylmethane dye Malachite Green. The decolorization reaction system included 50 mM sodium acetate buffer (pH 4.5), 20 mg / L dye, 6 mM MnCl2, and 0.9 U / mL enzyme solution (without enzyme as a control). 2 mM H2O2 was added to initiate the reaction, and the reaction was carried out at 25 °C for 12 h. The decolorization rate was then detected.

[0044] Example 2: Decolorization by peroxidase RhDyPB

[0045] Based on Example 1, the pH of the decolorization reaction system in step (3) was changed to 5.0, while the other conditions remained unchanged, and the decolorization rate was detected.

[0046] Example 3: Decolorization with peroxidase RhDyPB

[0047] Based on Example 1, the pH of the decolorization reaction system in step (3) was changed to 6.0, the reaction buffer was changed to 50mM sodium malonate buffer, and the other conditions remained unchanged. The decolorization rate was then detected.

[0048] Example 4: Changing enzyme and dye concentrations

[0049] Based on Example 3, the enzyme concentration in step (3) was changed to 1.8 U / mL, the dye concentration to 60 mg / L, and other conditions remained unchanged. The decolorization rate of malachite green was then measured. The results showed that the decolorization rate of malachite green was 81.3%.

[0050] Comparative Example 1: Changing the concentration of manganese ions

[0051] Based on Example 3, the amount of MnCl2 added in step (3) was changed to 2 mM, while other conditions remained unchanged, and the decolorization rate was detected. The results are as follows: Figure 1 As shown, the results indicate that reducing the manganese ion concentration resulted in a poorer decolorization effect on the anthraquinone dye Reactive Blue 19 and the oxane dye Eosin Y.

[0052] Comparative Example 2: Changing the concentrations of manganese ions and H2O2

[0053] Based on Example 3, the amount of MnCl2 added in step (3) was changed to 2mM, the concentration of H2O2 was changed to 0.1mM, and the other conditions remained unchanged. The decolorization rate was then detected.

[0054] Table 1 Enzyme Decolorization Rate Detection

[0055] Reactive Blue 19 Dawn Red Y Indigo Carmine Malachite Green Example 1 62.3％ 75.4％ 76.6％ 41.5％ Example 2 43.7％ 14.3％ 85.8％ 25.9％ Example 3 27.1％ - 86.2％ 94.4％ Comparative Example 1 23.60％ 2％ 95.5％ 75.30％ Comparative Example 2 40％ - 28.5％ 77.90％

[0056] Table 1 shows that RhDyPB exhibits varying decolorization effects on different dyes at different pH levels. With increasing pH, the decolorization rates of RhDyPB for the anthraquinone dye Reactive Blue 19 and the xanthene dye Eosin Y decrease; while the effects on the indigo dye Indigo Carmine and the triphenylmethane dye Malachite are better at higher pH levels. Specifically, at pH 4.5, RhDyPB achieves the highest decolorization rates for the anthraquinone dye Reactive Blue 19 and the xanthene dye Eosin Y, reaching 62.3% and 75.4%, respectively; at pH 6.0, RhDyPB achieves the highest decolorization rates for the indigo dye Indigo Carmine and the triphenylmethane dye Malachite, reaching 86.2% and 94.4%, respectively.

[0057] Reducing the manganese ion concentration decreases the decolorization rate of RhDyPB on three dyes: anthraquinone dye Reactive Blue 19, xanthracene dye Eosin Y, and triphenylmethane dye Malachite. At the same time, reducing the manganese ion concentration and H2O2 concentration decreases the decolorization rate of RhDyPB on all four dyes.

[0058] Example 5: Whole-cell dye decolorization

[0059] A method for whole-cell decolorization includes the following steps:

[0060] (1) Preparation of recombinant bacteria

[0061] The recombinant strain Eco / pE24AD constructed in Example 1 was used as a blank control, and the recombinant strain Eco / pE24b constructed with plasmid pET-24b was used as a blank control. After induction of expression, the bacterial cells were collected by centrifugation. The steps were the same as step (2) in Example 1.

[0062] (2) Take the bacterial cells prepared in step (1) and adjust them to OD values. 600 5. Eco / pE24AD cells were obtained by centrifugation of 3 mL of bacterial culture. The enzyme activity of the cells was 2.2 U / mL RhDyPB. These cells were used to decolorize anthraquinone dye Reactive Blue 19, oxanthracene dye Eosin Y, indigo dye Indigo Carmine, and triphenylmethane dye Malachite Green. The decolorization reaction system consisted of 50 mM sodium acetate buffer (pH 4.5), 20 mg / L dye, 6 mM MnCl2, and 5% (v / v) Tween-80. 2 mM H2O2 was added to initiate the reaction, which was carried out at 25°C for 12 h. An equal amount of Eco / pE24b cells was used as a control. The decolorization rate was measured after the reaction.

[0063] Example 6: Whole-cell decolorization

[0064] Based on Example 5, the pH of the decolorization reaction system was changed to 5.0, and the decolorization rate was detected after the reaction was completed.

[0065] Example 7: Whole-cell decolorization

[0066] Based on Example 5, the pH of the decolorization reaction system was changed to 6.0, and 50mM sodium malonate buffer was used. The decolorization rate was measured after the reaction was completed.

[0067] The dye decolorization rates of Examples 5-7 are shown in Table 2.

[0068] Table 2 Whole-cell decolorization rate

[0069] Reactive Blue 19 Dawn Red Y Indigo Carmine Malachite Green Eco / pE24b (Example 5) 1.8％ 21.2％ 51.7％ 19.3％ Eco / pE24AD (Example 5) 3％ 64.4％ 60.5％ 22％ Eco / pE24b (Example 6) 1.8％ 72.8％ 60％ 20.6％ Eco / pE24AD (Example 6) 8.4％ 75％ 81.5％ 32.5％ Eco / pE24b (Example 7) 15.3％ 94.5％ 83.3％ 30.2％ Eco / pE24AD (Example 7) 33％ 95％ 92.4％ 50.8％

[0070] The results in Table 2 show that, unlike enzyme catalysis, whole-cell decolorization efficiency increases with increasing pH for all dyes, and cells expressing RhDyPB show better decolorization than cells not expressing RhDyPB.

[0071] Meanwhile, whole-cell decolorization showed better decolorization effects than enzyme decolorization for the xanthracene dye Eosin Y and the indigo dye Indigo Carmine, with decolorization rates reaching 95% and 92.4% respectively at pH 6.0. However, the decolorization effect was not as good as enzyme-catalyzed decolorization for the anthraquinone dye Reactive Blue 19 and the triphenylmethane dye Malachite Green, with the highest decolorization rates being 33% and 50.8% respectively.

[0072] Comparative Example 4: Without Tween-80

[0073] Based on Example 5, Tween-80 was not added in step (3), and the decolorization rate was measured. The results showed that when Tween-80 was not added, the decolorization of whole-cell dyes was similar between the control group and the experimental group, and the RhDyPB effect in whole cells could not be fully utilized.

[0074] 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 method for decolorizing dyes using whole cells, characterized in that, The steps include: adding bacterial cells expressing dye decolorizing peroxidase to a decolorization reaction system, which consists of 2-6 mM MnCl2, 0.1-2 mM H2O2, and 0.5-5% (v / v) Tween-80; the pH of the decolorization system is 4.5-6.

0.

2. The method according to claim 1, characterized in that, The dyes are anthraquinone dye Reactive Blue 19, oxane dye Eosin Y, indigo dye Indigo Carmine, or triphenylmethane dye Malachite Green.

3. The method according to claim 1, characterized in that, The decolorization reaction system uses sodium acetate or sodium malonate at a concentration of 45–50 mM as a buffer solution.

4. The method according to claim 1, characterized in that, The bacterial enzyme activity in the decolorization system was 2.2~3.0 U / mL.

5. The method according to claim 1, characterized in that, The dye concentration in the decolorization reaction system is 20–60 mg / L.

6. The method according to claim 1, characterized in that, The decolorization reaction was carried out at 20–25°C for 10–24 hours.

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