Efficient hexavalent chromium reductase mutant and application thereof in chromium-containing wastewater
By using site-directed mutagenesis and immobilization techniques, a highly efficient hexavalent chromium reductase mutant was developed. Combined with a chitosan-nano Fe3O4 composite carrier, the problems of low catalytic efficiency and high cost in enzymatic treatment of chromium-containing wastewater were solved, enabling efficient and low-cost industrial application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CENT SOUTH UNIV
- Filing Date
- 2025-10-21
- Publication Date
- 2026-05-29
AI Technical Summary
Existing enzymatic methods for treating chromium-containing wastewater suffer from insufficient catalytic efficiency, easy enzyme deactivation, and difficulty in recycling and reuse, resulting in high treatment costs and failing to meet industrial needs.
A highly efficient hexavalent chromium reductase mutant was obtained through site-directed mutagenesis and immobilized using a chitosan-nano Fe3O4 composite carrier. Combined with an electron donor-assisted system, a composite hexavalent chromium catalytic system was formed for the treatment of chromium-containing wastewater.
The mutant exhibits a catalytic efficiency increase of 66.3%-92.1%, an enzyme half-life extended to 8.0 days, and a treatment rate of 99.77%, significantly reducing costs and adapting to complex industrial wastewater conditions.
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Figure CN122104612A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of environmental biology technology, specifically relating to a highly efficient hexavalent chromium reductase mutant and its application in chromium-containing wastewater. Background Technology
[0002] Chromium (Cr) pollution in soil and groundwater is one of the most concerning environmental issues internationally, primarily originating from industries such as chromium salt production, electroplating, tanning, pigment production, mining, and metallurgy. Chromium exists in two stable oxidation states in the environment: hexavalent chromium [Cr(VI)] and trivalent chromium [Cr(III)]. Cr(VI) is non-biodegradable and readily absorbed by the human body, entering organs through air, drinking water, food, or skin contact, causing symptoms such as nasal mucosal atrophy, vomiting, diarrhea, and allergic dermatitis. Long-term exposure may even pose a carcinogenic risk. In contrast, Cr(III) has low solubility, weak migration ability, and low biotoxicity; therefore, reducing Cr(VI) to Cr(III) has become the core strategy for chromium pollution control.
[0003] Existing methods for treating chromium-containing wastewater include chemical precipitation, ion exchange resin methods, adsorption, and biological methods. Chemical precipitation is suitable for high-concentration wastewater but is prone to secondary pollution; ion exchange resin methods suffer from high investment costs and poor selectivity; adsorption and biological methods are suitable for low-concentration wastewater, but have problems with long treatment cycles and unsatisfactory removal rates. Enzymatic treatment has attracted attention due to its advantages of being green, having no secondary pollution, high selectivity, and high efficiency. Patents have reported the application of hexavalent chromium reductase, such as the enzyme preparation disclosed in the patent "An Old Yellow Enzyme OYE2 Protein and Its Application in Chromium Pollution," which showed a removal rate of only 38% after 6 hours under specific conditions. Existing enzyme preparations generally suffer from insufficient catalytic efficiency and limited tolerance to environmental conditions, making it difficult to meet industrial needs.
[0004] Meanwhile, free enzymes are prone to inactivation and difficult to recover and reuse in industrial applications, resulting in high treatment costs. This has become a major bottleneck for the large-scale application of enzymatic treatment technology, and enzyme immobilization is a common method to address this problem. Therefore, developing highly active enzyme mutants and combining them with immobilization technology is crucial for promoting the industrialization of enzymatic treatment of chromium-containing wastewater. Summary of the Invention
[0005] The purpose of this invention is to provide a highly efficient hexavalent chromium reductase mutant and its application in chromium-containing wastewater, so as to solve the problems mentioned in the background art.
[0006] To achieve the above objectives, the present invention provides the following technical solution:
[0007] A hexavalent chromium reductase mutant, wherein the mutant is obtained by site-directed mutagenesis using the wild-type hexavalent chromium reductase SOYE shown in SEQ ID NO:1 as the parent, includes the following three mutants:
[0008] Mutant 1: The amino acid sequence of the mutant is modified from the wild type by C25G / I71G / D75M / V159I / D260A / P298G / G347A mutations.
[0009] Mutant 2: The amino acid sequence of the mutant is modified from the wild type by the C25G / I71G / D75M / V159I / D260A / P298G / A310P / G347A mutation.
[0010] Mutant 3: The amino acid sequence of the mutant is modified from the wild type by the mutations C25G / I71G / D75M / V159I / S196P / D260A / P298G / A310P / G347A.
[0011] Preferably, the nucleotide sequence of the hexavalent chromium reductase mutant is as follows:
[0012] SEQ ID NO:1
[0013] Wild-type amino acid sequence
[0014] MSALFEPYALRDLTIPNRVWMAPMCQYSAAPEGPDTGAANDWHFAHYAARATGGTGLILVEATAVHPDGRITPYDLGIWNDRQVEALRRITDFLRTQGTVPGIQLAHAGRKGSTGRPWEGGGAVGPDAHGW QPVAPSAIAFAEGHPVPSELTVDEIRAVVRQFADAARRALAAGFEVAEIHGAHGYLINEFLSPHSNHRTDEYGGSYENRTRLALEVVDAVRAVWPQDKPLFFRISATDWLGEDGWTDDDTVRLAADLHDHGV
[0015] DLLDVSTGGNSPSARIP
[0016] VGPGYQVPFAARVKAETPLPVAAVGLITDAEQAEKILANGEADAILLGRELLRNPSFARHAARELGGEVRVPDQYHRSV
[0017] SEQ ID NO:2
[0018] Wild-type nucleotide sequence
[0019]
[0020] SEQ ID NO:3
[0021] >Optimized wild-type nucleotide sequence
[0022]
[0023] Preferably, a recombinant expression vector contains a nucleotide sequence, and the expression vector is a pET-28a(+) vector.
[0024] Preferably, a recombinant strain contains a recombinant expression vector, and the host cell is E. coli BL21(DE3).
[0025] Preferably, an immobilized hexavalent chromium reductase preparation is prepared by immobilizing mutant 3 as the active ingredient using a chitosan-nano Fe3O4 composite carrier. The specific immobilization steps are as follows: chitosan is dissolved in 1% acetic acid solution, nano Fe3O4 is added and ultrasonically dispersed, the pH is adjusted to 6.0, glutaraldehyde is added for cross-linking, then pure enzyme solution of mutant 3 is added, the mixture is shaken and adsorbed at 25°C for 4 hours, and the product is collected by magnetic separation.
[0026] Preferably, a composite hexavalent chromium catalytic system comprises an immobilized hexavalent chromium reductase preparation and an electron donor auxiliary system; the electron donor auxiliary system is composed of NADH at a final concentration of 0.5-10 mM; and the amount of the immobilized enzyme preparation added to the system is 0.15-0.25 mg / mL.
[0027] Preferred applications include the use of hexavalent chromium reductase mutants, immobilized hexavalent chromium reductase preparations, or composite catalytic systems in the treatment of chromium-containing wastewater.
[0028] Preferably, the application method is as follows: a composite catalytic system is added to the chromium-containing wastewater, and the reaction is carried out at a temperature of 15-40℃ and a pH of 6.0-8.0 for 10-120 minutes; the Cr(VI) concentration in the chromium-containing wastewater is 0.1mM-4.0mM.
[0029] Preferably, the amount of immobilized enzyme preparation added in the composite catalytic system is 0.2 mg / mL, the final concentration of NADH in the electron donor auxiliary system is 10 mM, the concentration of hexavalent chromium is 200 mg / L, and the reaction conditions are 30℃ and pH 7.0.
[0030] Preferably, after the reaction is completed, the immobilized enzyme preparation is recovered by magnetic separation and can be reused after washing with 0.01M PBS buffer.
[0031] Compared with the prior art, the present invention provides a highly efficient hexavalent chromium reductase mutant and its application in chromium-containing wastewater, which has the following beneficial effects:
[0032] Through multi-site combined mutations, the catalytic efficiency of the three mutants was higher than that of the wild type (1.472 mM⁻¹). s -1) The values were increased by 66.3%, 74.5%, and 92.1% respectively, with mutant 3 achieving a kcat / Km value of 2.827 mM-1.s It has a -1 affinity for NADH and significantly improves the affinity for NADH, which to some extent solves the problem of insufficient activity of existing enzyme preparations.
[0033] The use of chitosan-nano Fe3O4 composite carrier extends the half-life of enzymes from 2.5 days in the free state to 8.0 days, and can be quickly recovered through magnetic separation. It can be reused 8 times and still maintain high activity, which greatly reduces the cost of enzyme preparation consumption.
[0034] The composite hexavalent chromium catalytic system has a wide range of temperature and pH adaptability (15-40℃, pH 6.0-8.0), making it suitable for the complex water quality conditions of industrial wastewater.
[0035] The treatment rate of actual industrial wastewater reaches 99.77%, the effluent meets the standards, and the treatment cycle is only 2 hours. Combined with the reusability of immobilized enzymes, the cost threshold for industrial application is significantly reduced. Attached Figure Description
[0036] Figure 1 This invention demonstrates the electrophoretic analysis of wild-type and mutant proteins; the symbols from right to left are: M: protein marker; WT: wild-type; 3: C25G / I71G / D75M / V159I / D260A / P298G / G347A; 4: C25G / I71G / D75M / V159I / D260A / P298G / A310P / G347A; 5: C25G / I71G / D75M / V159I / S196P / D260A / P298G / A310P / G347A.
[0037] Figure 2 This invention demonstrates the effect of temperature on the removal efficiency of Cr(VI) in wild-type and mutant organisms.
[0038] Figure 3 This invention demonstrates the effect of pH on the removal efficiency of Cr(VI) in wild-type and mutant organisms.
[0039] Figure 4 This invention demonstrates the reduction effect of hexavalent chromium reductase on Cr(VI)-containing wastewater. Detailed Implementation
[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0041] This invention provides, for example Figure 1-4 shown
[0042] A hexavalent chromium reductase mutant, obtained by site-directed mutagenesis using the wild-type hexavalent chromium reductase SOYE shown in SEQ ID NO:1 as the parent, includes the following three mutants:
[0043] Mutant 1: The amino acid sequence of the mutant is modified from the wild type by C25G / I71G / D75M / V159I / D260A / P298G / G347A mutations.
[0044] Mutant 2: The amino acid sequence of the mutant is modified from the wild type by the C25G / I71G / D75M / V159I / D260A / P298G / A310P / G347A mutation.
[0045] Mutant 3: The amino acid sequence of the mutant is modified from the wild type by the mutations C25G / I71G / D75M / V159I / S196P / D260A / P298G / A310P / G347A.
[0046] The nucleotide sequence of the hexavalent chromium reductase mutant is as follows:
[0047] SEQ ID NO:1
[0048] Wild-type amino acid sequence
[0049] MSALFEPYALRDLTIPNRVWMAPMCQYSAAPEGPDTGAANDWHFAHYAARATGGTGLILVEATAVHPDGRITPYDLGIWNDRQVEALRRITDFLRTQGTVPGIQLAHAGRKGSTGRPWEGGGAVGPDAHGW QPVAPSAIAFAEGHPVPSELTVDEIRAVVRQFADAARRALAAGFEVAEIHGAHGYLINEFLSPHSNHRTDEYGGSYENRTRLALEVVDAVRAVWPQDKPLFFRISATDWLGEDGWTDDDTVRLAADLHDHGV
[0050] DLLDVSTGGNSPSARIP
[0051] VGPGYQVPFAARVKAETPLPVAAVGLITDAEQAEKILANGEADAILLGRELLRNPSFARHAARELGGEVRVPDQYHRSV
[0052] SEQ ID NO:2
[0053] Wild-type nucleotide sequence
[0054]
[0055] SEQ ID NO:3
[0056] >Optimized wild-type nucleotide sequence
[0057]
[0058] A recombinant expression vector containing a nucleotide sequence, wherein the expression vector is a pET-28a(+) vector.
[0059] A recombinant bacterial strain containing a recombinant expression vector, with E. coli BL21(DE3) as the host cell.
[0060] An immobilized hexavalent chromium reductase preparation, using mutant 3 as the active ingredient, is prepared by immobilization using a chitosan-nano Fe3O4 composite carrier. The specific immobilization steps are as follows: chitosan is dissolved in 1% acetic acid solution, nano Fe3O4 is added and ultrasonically dispersed, the pH is adjusted to 6.0, glutaraldehyde is added for cross-linking, then pure enzyme solution of mutant 3 is added, the mixture is shaken and adsorbed at 25°C for 4 hours, and the product is collected by magnetic separation.
[0061] A composite hexavalent chromium catalytic system comprises an immobilized hexavalent chromium reductase preparation and an electron donor auxiliary system; the electron donor auxiliary system consists of NADH at a final concentration of 0.5-10 mM; the amount of the immobilized enzyme preparation added to the system is 0.15-0.25 mg / mL.
[0062] Application of hexavalent chromium reductase mutants, immobilized hexavalent chromium reductase preparations, or composite catalytic systems in the treatment of chromium-containing wastewater; the specific application method is as follows: add the composite catalytic system to the chromium-containing wastewater and react for 10 min to 120 min at a temperature of 15-40℃ and a pH of 6.0-8.0; the Cr(VI) concentration in the chromium-containing wastewater is 0.1 mM to 4.0 mM.
[0063] The amount of immobilized enzyme preparation added in the composite catalytic system was 0.2 mg / mL. The final concentration of NADH in the electron donor auxiliary system was 10 mM, and the concentration of hexavalent chromium was 200 mg / L. The reaction conditions were 30℃ and pH 7.0.
[0064] After the reaction, the immobilized enzyme preparation was recovered by magnetic separation and could be reused after washing with 0.01M PBS buffer.
[0065] Example 1: Construction of a hexavalent chromium reductase mutant
[0066] Wild-type gene cloning: Using Streptomycessp.M7 genomic DNA as a template, primer pairs were designed (upstream: 5'-GTGAGTGCGCTCTTCGAGCCCTAC-3'; downstream: 5'-ACCGGTACCCTCGAGTTACACCGGTACC-3'), and the wild-type nucleotide sequence shown in SEQ ID NO:2 was obtained by PCR amplification. The sequence was cloned into the pET-28a(+) vector and transformed into E. coli BL21(DE3).
[0067] Construction of a randomized mutant library: The SOYE gene was amplified using error-prone PCR. The reaction system contained 0.2 mM dATP / dGTP, 1 mM dCTP / dTTP, and 0.5 mM MnCl2. The amplified product was ligated into a vector and transformed into host bacteria to construct a library containing 10... 4 A cloned random mutant library.
[0068] Site-directed mutagenesis: For the activity-enhancing mutants obtained through screening, site-directed mutagenesis primers (such as S196P mutation primer: 5'-GCGGCTCGTACGAGAACCGCACCCGTCTCCCGCTCGAGGTC-3') are designed, and the mutation sites are introduced through overlap extension PCR to construct combinatorial mutants.
[0069] Screening and validation: Single clones were selected, cultured, and induced to express. Crude enzyme solution was used for Cr(VI) reduction reaction. The concentration of Cr(VI) was determined by diphenylcarbazide spectrophotometry, the removal rate was calculated, and three highly active mutants were identified by combining SDS-PAGE and kinetic parameter determination.
[0070] Example 2: Expression and purification of mutants
[0071] Seed culture: Select a single colony of the recombinant strain containing mutant gene 3, inoculate it into LB medium containing 50 μg / mL kana, and incubate at 37℃ and 200 rpm for 12 h.
[0072] Expanded culture and induction: Transferred to TB medium containing 50 μg / mL kana at a ratio of 1:100, cultured at 37℃ until OD600=0.9, added IPTG to a final concentration of 0.25mM, and induced at 25℃ for 6h.
[0073] Protein purification: Collect bacterial cells by centrifugation at 8000 rpm for 10 min, resuspend in PBS buffer, add lysozyme (final concentration 1 mg / mL), incubate on ice for 30 min, and sonicate (300 W, 3 seconds on, 5 seconds off, total 30 min). Centrifuge at 12000 rpm for 20 min and collect the supernatant to obtain crude enzyme solution. Purify using a Ni-NTA affinity chromatography column. Wash with buffer containing 20 mM imidazole, elute with 500 mM imidazole buffer, collect the eluent, and dialyze to desalt to obtain pure enzyme solution.
[0074] Example 3: Preparation of immobilized enzyme preparations
[0075] Preparation of composite carrier: Weigh 2g of chitosan, dissolve it in 100mL of 1% acetic acid solution, stir until completely dissolved, add 0.4g of nano Fe3O4, ultrasonically disperse for 20min, slowly add 1M NaOH to adjust the pH to 6.0, add 1mL of glutaraldehyde, and crosslink at 30℃ with constant temperature and shaking for 1h.
[0076] Enzyme immobilization: 10 mL of mutant 3 pure enzyme solution (concentration 1 mg / mL) was added to the cross-linked carrier system, and the mixture was shaken at 25 °C and 150 rpm for 4 h to adsorb the enzyme. The carrier was collected by adsorption with a magnet and washed 3 times with 10 mL of 0.01 M PBS buffer (pH 7.0) each time to obtain the immobilized enzyme preparation. The enzyme activity recovery rate reached 82%.
[0077] Example 4: Performance determination of the composite catalytic system
[0078] Temperature tolerance experiment: At different temperatures (15℃, 20℃, 25℃, 30℃, 35℃, 40℃), the composite system (immobilized enzyme dosage of 0.2 mg / mL, final NADH concentration of 10 mM) was added to a reaction solution with a Cr(VI) concentration of 200 mg / L, and the reaction was carried out at pH 7.0 for 2 h. The removal rate was then measured. The results showed that the activity was highest at 30℃, and it still maintained more than 85% of the highest activity at 15℃ and 40℃. Figure 2 ).
[0079] pH tolerance experiment: The reaction was carried out at 30℃ for 2 hours under different pH conditions (6.0, 6.5, 7.0, 7.5, 8.0), and the removal rate was measured. The results showed that the activity was highest at pH 7.0, and it still maintained more than 88% of the highest activity at pH 6.0 and 8.0. Figure 3 ).
[0080] Reusability experiment: The reaction was carried out under optimal conditions. After each reaction, the immobilized enzyme was magnetically separated and recovered, washed, and reused. The removal rate of each reaction was measured. The results showed that after 8 cycles, the removal rate still reached 70.2%.
[0081] Example 5: Application in Chromium-Containing Wastewater Treatment
[0082] Simulated wastewater treatment: Prepare simulated wastewater with a Cr(VI) concentration of 200 mg / L (containing 0.5% NaCl, pH 7.0). Take 100 mL of the wastewater and add it to the composite catalytic system. React at 30℃ and 150 rpm for 2 hours with shaking. Take a sample to determine the Cr(VI) concentration. The calculated removal rate is 98.3%.
[0083] Actual wastewater treatment: 100 mL of chromium-containing wastewater (Cr(VI) concentration 185 mg / L, pH after neutralization approximately 7, COD 320 mg / L) from an electroplating enterprise in Hunan Province was taken and added to a composite catalytic system. The mixture was reacted at 30°C and 150 rpm for 2 hours. The Cr(VI) concentration was measured to be reduced to 0.42 mg / L, with a removal rate of 99.77%, which meets the requirements of the "Electroplating Pollutant Discharge Standard" (GB21900-2008).
[0084] The catalytic efficiencies of the wild type and mutant are shown in the table below:
[0085]
[0086]
[0087] Table 1
[0088] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.