CN113735282B - Old yellow enzyme OYE2 protein and application thereof in chromium pollution - Google Patents
Old yellow enzyme OYE2 protein and application thereof in chromium pollution Download PDFInfo
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- CN113735282B CN113735282B CN202111103840.7A CN202111103840A CN113735282B CN 113735282 B CN113735282 B CN 113735282B CN 202111103840 A CN202111103840 A CN 202111103840A CN 113735282 B CN113735282 B CN 113735282B
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/20—Heavy metals or heavy metal compounds
- C02F2101/22—Chromium or chromium compounds, e.g. chromates
Abstract
The invention discloses a yellow-light enzyme OYE2 protein and application thereof in chromium pollution, belonging to the field of heavy metal prevention and treatment. The invention takes old yellow enzyme OYE2 protein in Corynebacterium crenatum SYPA5-5 as an effective component for reducing Cr (VI), has activity of hexavalent chromium reductase, has stronger reducing effect on Cr (VI), and can quickly remove Cr (VI) in wastewater. By adopting the method, the concentration of Cr (VI) in the wastewater without the hexavalent chromium reductase is not obviously changed, the concentration of Cr (VI) in the wastewater added with the hexavalent chromium reductase is reduced to 62mg/L, the reduction rate reaches 38 percent, and the hexavalent chromium in the wastewater can be quickly and effectively removed. Therefore, the hexavalent chromium reductase prepared by one or more of the enzymes has the potential of industrial application in treating the Cr (VI) containing wastewater.
Description
Technical Field
The invention relates to a yellow-light enzyme OYE2 protein and application thereof in chromium pollution, belonging to the field of heavy metal prevention and treatment.
Background
Chromium (Cr) pollution in soil and groundwater is one of the most internationally interesting environmental problems, mainly originating from industries such as chromium salt production, electroplating, tanning, pigment production, mining and metallurgy. Chromium exists in two stable chromium oxidation states in the environment: hexavalent chromium and trivalent chromium; trivalent chromium Cr (III) has low solubility, poor migration ability and low biotoxicity compared to hexavalent chromium Cr (VI). Therefore, one of the effective strategies for treating chromium pollution in water is to reduce hexavalent chromium Cr (VI) to trivalent chromium Cr (III). The reduction of hexavalent chromium Cr (VI) by microorganisms is considered to be a method with great application potential, but the microbial remediation process still has the defect of long period, and the in-situ remediation by microorganisms is greatly influenced by environmental factors and competes with indigenous microorganisms, so that the remediation effect is unstable.
At present, a large number of researches report that hexavalent chromium reductase is used for repairing soil and treating polluted underground water, such as repairing DDT (dichloro-diphenyl-trichloroethane) -polluted soil by using laccase, repairing lead-polluted soil by using soil enzyme, repairing pesticide-polluted soil by using immobilized enzyme, performing denitrification treatment on underground water by using enzyme generated by nitrobacteria, and the like. For Cr (VI) contamination, there have been numerous studies reporting Cr (VI) reductases, such as NfsA and YieF enzymes from Escherichia coli, with maximum reduction rates of 0.25 and 5.0. Mu. Mol/min/mg, respectively; the maximum reduction rate of ChrR in Pseudomonas putida KT2400 was 8.8. Mu. Mol/min/mg. The proteases can realize the effective reduction of Cr (VI) under certain conditions, but have a large gap from large-scale industrial application. Therefore, a method for efficiently treating chromium pollution in water is urgently needed to meet large-scale industrial application.
Disclosure of Invention
The invention provides hexavalent chromium reductase and application thereof in treating chromium pollution of a water body, in order to obtain a method for efficiently treating chromium pollution in the water body. The application is to put the hexavalent chromium reductase into a hexavalent chromium polluted water body, reduce the highly water-soluble hexavalent chromium into the low water-soluble trivalent chromium, and achieve the treatment purpose by reducing the mobility of the low water-soluble trivalent chromium.
The invention provides a method for degrading hexavalent chromium by an enzyme method, which is characterized in that hexavalent chromium reductase with an amino acid sequence shown as SEQ ID NO.1 is added into a reaction system containing hexavalent chromium for degradation.
In one embodiment of the invention, the nucleotide sequence encoding the hexavalent chromium reductase is set forth in SEQ ID No. 2. The result of the injection in GenBank is riboflavin oxidoreductase/NADH oxidase, the protein belongs to OYE2 family members, and the invention names the protein Cyc1.
In one embodiment of the invention, the hexavalent chromium reductase is derived from Corynebacterium crenatum SYPA5-5.
In one embodiment of the present invention, the amount of hexavalent chromium reductase added to the reaction system is at least 0.1U/mg hexavalent chromium Cr (VI).
In one embodiment of the invention, the hexavalent chromium reductase is dosed in an amount of at least 0.1U/mg hexavalent chromium Cr (VI).
In one embodiment of the present invention, when the chromium concentration in the polluted water body is less than or equal to 100mg/L, the dosage of the hexavalent chromium reductase is 0.1U/mg Cr (VI). Converted from kinetic maximum velocity Vmax =30.39umol Cr (VI)/min/mg enzyme protein.
In one embodiment of the present invention, the degradation reaction conditions are: is carried out under the condition of pH 6-12.
In one embodiment of the invention, the optimum working pH of the enzyme is 8.5.
In one embodiment of the invention, the degradation reaction is carried out at a temperature of 25-40 ℃.
In one embodiment of the present invention, the hexavalent chromium reductase has an optimum working temperature of 30 ℃.
The invention also provides a recombinant escherichia coli, which contains the hexavalent chromium reductase of which the amino acid sequence is shown in SEQ ID No. 1.
In one embodiment of the invention, the nucleotide sequence encoding the hexavalent chromium reductase is set forth in SEQ ID No. 2.
In one embodiment of the invention, the recombinant escherichia coli takes e.coli BL21 (DE 3) as an expression host and takes pXMJ-19 as an expression vector.
The invention also provides a method for degrading hexavalent chromium in a whole cell manner, which is characterized in that microbial cells containing hexavalent chromium reductase with an amino acid sequence shown as SEQ ID NO.1 are added into a reaction system containing hexavalent chromium for degradation.
In one embodiment of the invention, the reaction system is a sewage environment.
In one embodiment of the invention, the nucleotide sequence encoding the hexavalent chromium reductase is set forth in SEQ ID No. 2.
In one embodiment of the invention, the microbial cell is a recombinant E.coli.
In one embodiment of the invention, the recombinant escherichia coli takes e.coli BL21 (DE 3) as an expression host and takes pXMJ-19 as an expression vector.
In one embodiment of the present invention, the recombinant E.coli is added in an amount of at least 1% (v/v) in the reaction system.
The invention also provides a method for improving the degradation capability of the escherichia coli on hexavalent chromium, which is to express the hexavalent chromium reductase with an amino acid sequence shown as SEQ ID NO.1 in the escherichia coli.
In one embodiment of the invention, the nucleotide sequence encoding the hexavalent chromium reductase is set forth in SEQ ID No. 2.
The invention also provides application of the hexavalent chromium reductase with an amino acid sequence shown as SEQ ID NO.1, or a recombinant vector containing the hexavalent chromium reductase with a nucleotide sequence shown as SEQ ID NO.2, or a microbial cell containing the hexavalent chromium reductase with an amino acid sequence shown as SEQ ID NO.1 in preparation of a product for degrading hexavalent chromium.
In one embodiment of the invention, the recombinant vector is pET-28a, pDXW-10 and pXMJ-19 as an expression vector.
In one embodiment of the present invention, the microbial cell is a bacterial or fungal host cell.
In one embodiment of the invention, the microbial cells are E.coli BL21, E.coli JM109, corynebacterium crenatum SYPA5-5 or Corynebacterium ATCC 13032 as host cells.
In one embodiment of the invention, the product is a chemical.
The invention also provides a preparation method of the hexavalent chromium reductase capable of quickly removing Cr (VI) in the wastewater, the hexavalent chromium reductase gene Cyc1 is constructed on an expression vector, and a prokaryotic expression system is utilized to prepare the high-activity chromium reductase Cyc1.
In one embodiment of the invention, a Corynebacterium crenatum SYPA5-5 genome is used as a template, the following primers are designed, a nucleic acid sequence (shown as SEQ ID NO. 2) of a chromium reductase gene Cyc1 is obtained through PCR amplification, the nucleic acid sequence is constructed on a pXMJ-19 vector, BL21 large intestinal rod competent cells are transformed, and the target protein is obtained through induced expression, separation and purification of the obtained recombinant bacteria. The primer sequences involved are as follows:
F：5’-AGCTTAAAGGAGGACAACCGTGCACCACCACCACCACCACCTCCAAGCTGT-3’
R：5’-AGCCAAGTGAATTCTTAAAGTACGTAGTC-3’。
advantageous effects
(1) The chromium reductase from Corynebacterium crenatum SYPA5-5 provided by the invention has stronger reducing effect on Cr (VI) and can rapidly remove Cr (VI) in wastewater.
(2) By adopting the method, the concentration of Cr (VI) in the wastewater without the hexavalent chromium reductase is not obviously changed, the concentration of Cr (VI) in the wastewater added with the hexavalent chromium reductase is reduced to 62mg/L, the reduction rate reaches 38 percent, and the hexavalent chromium in the wastewater can be quickly and effectively removed. Therefore, the hexavalent chromium reductase prepared by one or more of the enzymes has industrial application potential in treating Cr (VI) containing wastewater.
Drawings
FIG. 1: the electrophoresis pattern of the PCR amplification result of the Cr (VI) reducing gene Cyc1 in the embodiment 1 of the invention; wherein, M: a nucleic acid Marker; lanes 1, 2, 3 and 4: cyc1PCR amplified product.
FIG. 2: the expression vector pXMJ-19 in the embodiment 1 of the invention is subjected to double enzyme digestion to obtain an electrophoresis picture; wherein, M: a nucleic acid Marker; lanes 1, 2: pXMJ-19 was double digested (EcoR I and Hind III).
FIG. 3: SDS-PAGE picture of the crude enzyme liquid of the chromium reductase Cyc1 in the embodiment 2 of the invention; wherein, M: protein Marker; lane 1: pXMJ19 empty control; lane 2: inducing Cyc1 protein expression at 15 ℃; lane 3: inducing Cyc2 protein expression at 20 ℃; lane 4: inducing Cyc1 protein expression at 25 ℃; lane 5: cyc1 protein expression was induced at 30 ℃.
FIG. 4 is a schematic view of: SDS-PAGE picture after purifying chromium reductase Cyc1 in example 2 of the invention; wherein, M: a protein Marker; lane 1: inducing Cyc1 protein expression at 30 ℃; lane 2: inducing Cyc2 protein expression at 25 ℃; lane 3: cyc1 protein expression was induced at 20 ℃.
FIG. 5: effect of temperature on hexavalent chromium reductase activity in example 3 of the present invention.
FIG. 6: the influence of the pH value on the activity of the hexavalent chromium reductase in example 3 of the present invention.
FIG. 7: the influence of different metal ions on the activity of the hexavalent chromium reductase in example 3 of the invention.
FIG. 8: the effect of different concentrations of Cr (VI) on the activity of hexavalent chromium reductase in example 3 of the present invention.
FIG. 9: the degradation condition of the recombinant bacteria in embodiment 3 of the invention to Cr (VI) is disclosed.
FIG. 10: the hexavalent chromium reductase in the embodiment 4 of the invention reduces the Cr (VI) containing wastewater.
Detailed Description
Corynebacterium crenatum SYPA5-5 is an L-arginine high-producing strain bred in the laboratory, corynebacterium crenatum SYPA5-5 is described in the patent application text with the publication number of CN1441055A, and the preservation number is CGMCC NO.0890 (the strain in the patent application text is SDNN403, and the inventor renumbers the strain as SYPA5-5 in the experimental process); based on pre-transcriptome and proteome data analyses, hexavalent chromium reductase (Cyc 1) was found to be up-regulated in expression levels under high versus low dissolved oxygen conditions. In order to further research the anti-oxidative stress function of Cyc1, an over-expression strain and a knockout bacterium of Cyc1 are constructed, and Cyc1 protein is found to have extremely strong redox property. Cyc1 is taken as a research object, the circular dichroism structure of Cyc1 is analyzed, SWISS-MODEL modeling analysis is used, the reduction capability of the Cyc1 on Cr (VI) is detected, and the structure and the function of Cyc1 are verified. The Cyc1 protein has a strong reduction effect on Cr (VI), can rapidly remove Cr (VI) in wastewater, and has an important application value in the treatment of wastewater containing Cr (VI).
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.
The media involved in the following examples are as follows:
LB liquid Medium (g/L): 5 yeast powder, 10 tryptone and 10 sodium chloride.
LB solid Medium: on the basis of LB liquid culture medium, 1.5-2.0% agar powder is added.
The detection methods referred to in the following examples are as follows:
detection of protein concentration:
protein concentration was determined using the Bradford kit with bovine serum albumin as standard.
Detection of Cr (VI) concentration:
in the dibenzoyl dihydrazide method, 0.3ml of a sample to be tested was sampled, 2.5ml of a 0.12M concentrated sulfuric acid solution was added, and 0.2ml of dibenzoyl dihydrazide (dissolved in acetone) was added to the above mixture so that the final concentration became 0.4mM, and the concentration was measured at an absorbance of 543 nm.
Methods for extracting genomic DNA and related plasmids referred to in the following examples:
when the genomic DNA is extracted from Corynebacterium crenatum SYPA5-5, the operation is carried out according to the instruction of the kit for rapidly extracting the genomic DNA of the Shanghai strap-down bacteria. When plasmid is extracted, bacterium liquid with proper concentration is cultured and centrifuged, supernatant is removed, corynebacterium glutamicum is gram-positive bacterium, when recombinant strains of the corynebacterium glutamicum is used for extracting plasmids, a proper amount of lysozyme is required to be added in water bath at 37 ℃ for 0.5h, when the recombinant strains of escherichia coli are used for extracting plasmids, lysozyme is not required to be added, a strapdown small plasmid extraction kit is used for extraction, and detailed operation reference instructions are provided.
Coli competence was prepared and chemically transformed as follows in the following examples:
coli transformed Competent cells were prepared using the Competent Cell Preparation Kit of Takara corporation, see the instruction manual for detailed operation. Transformation to E.coli BL21 is carried out by heat shock at 42 ℃, positive transformants are obtained by antibiotic resistance plate screening, plasmids are extracted for PCR verification and are sent to Jin Weizhi Biotech company for sequencing verification.
Example 1: construction of genetically engineered bacterium capable of expressing Cr (VI) reductase gene Cyc1
1. Cloning of Cyc1 Gene
The target gene (the nucleotide sequence is shown as SEQ ID NO. 2) is subjected to PCR amplification by adopting high fidelity enzyme PhantaR Max (p 515) DNA polymers, the PCR system is a standard reaction system of 50 mu L, and the PCR amplification program is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s; the annealing temperature is generally set to be 58-60 ℃ for 30-60 s; setting time for amplifying 1500bp genes at 72 ℃ per minute; denaturation to extension procedure for 30 cycles; further extension at 72 deg.C for 5min; storing at 4 ℃. After the PCR was completed, the PCR product was subjected to nucleic acid electrophoresis, and verified and gel recovered and purified, and the results are shown in FIG. 1. Carrying out double enzyme digestion with a linearized vector expression vector pXMJ-19 after recovery, wherein the result is shown in figure 2; e.coli BL21 (DE 3) competent cells were transformed after ligation using Cloneexpress II One Step Cloning Kit, and plasmids were extracted from positive transformants for PCR and sequencing.
Preparing the genetic engineering bacterium E.coli BL21 (DE 3)/pXMJ-19-Cyc 1 capable of expressing hexavalent chromium reductase Cyc1.
The empty pXMJ-19 plasmid was introduced into E.coli BL21 (DE 3) competent cells according to the method described above to prepare a control strain E.coli BL21 (DE 3)/pXMJ-19.
Example 2: expression and purification of hexavalent chromium reductase Cyc1 protein
1. Induced expression of hexavalent chromium reductase Cyc1 protein
The genetically engineered bacteria E.coli BL21 (DE 3)/pXMJ-19-Cyc 1 and E.coli BL21 (DE 3)/pXMJ-19 prepared in example 1 were streaked to contain Chl R After being activated at 37 ℃ for 12 hours on the LB solid medium, single colonies were picked up and inoculated into 10mL of solid medium containing Chl R Culturing the seeds in LB liquid culture medium vials at 30 ℃ and 180rpm for 16-24 h, and respectively preparing seed liquid;
the prepared seed solution was transferred to 4 bottles of 50mL containing Chl at an inoculum size of 1% (v/v) R After culturing the cells in the LB liquid medium at 30 ℃ and 180rpm for 4 to 5 hours, isopropyl beta-D-1-thiogalactoside (IPTG) is added to the cells until the final concentration is 1 mmol.L -1 Respectively carrying out induction expression for 16-18 h at 15 ℃,20 ℃, 25 ℃,30 ℃ and 180rpm, and then preparing the hair under different temperature inductionFermenting liquid;
respectively centrifuging the prepared fermentation liquor at 4 deg.C and 8000rpm for 10min to collect thallus cells.
Respectively collecting the cells with 0.1 mol. L -1 After washing twice with PBS buffer (pH 7.4), the cells were resuspended in PBS buffer (pH 7.4) and the cell concentration (OD: cell concentration) was controlled 600 = 6.0), crushing by using an ultrasonic crusher, centrifuging the crushed liquid for 20min at 4 ℃, collecting supernatant, analyzing the protein expression condition by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and obtaining a result shown in figure 3, wherein the induction expression effect of the hexavalent chromium reductase Cyc1 protein at 30 ℃ is the best as can be seen from figure 3; the residual crude enzyme solution is used for subsequent enzyme activity determination.
Preparing a crude enzyme solution containing hexavalent chromium reductase Cyc1 under different temperature induced expressions.
2. Purification of hexavalent chromium reductase Cyc1 protein
Respectively filtering the crude enzyme solution containing the hexavalent chromium reductase Cyc1 prepared in the step 1 by adopting a 0.45-micrometer filter membrane, purifying by adopting a Ni-NTA protein purification column to remove foreign proteins, wherein the tail end of the recombinase Cyc1 is provided with a his label capable of being matched with Ni on the purification column + Chelating, gradient eluting with imidazole buffer solution of different concentrations, eluting the purified protein bound on the purifying column, and further purifying the enzyme. Then, SDS-PAGE analysis is carried out on the Cyc1 pure enzyme solution, as shown in figure 4, and as can be seen from figure 4, the induction expression effect of the hexavalent chromium reductase Cyc1 protein is best at 30 ℃.
The concentration of the hexavalent chromium reductase Cyc1 protein under 30 ℃ induced expression is measured by using a Bradford kit, and bovine serum albumin is used as a standard substance.
Specific enzyme activity data of pure hexavalent chromium reductase Cyc1 enzyme in a buffer solution containing 20mM MOPS-NaOH comprises 100mM NAD (P) H, 10mM FMN and 1mM CrO 3 And 10mM CaCl 2 50. Mu.L of Cyc1 pure enzyme was added and reacted at 30 ℃ and pH =8.5 for 1min, and the Cr (VI) content was measured by the diphenylcarbodihydrazide method, and the results are shown in Table 1.
TABLE 1 specific enzyme activity (U/mg) of Cyc1 pure enzyme solution on Cr (VI) reduction under different ligands
Example 3: activity analysis of pure enzyme of hexavalent chromium reductase Cyc1
The following experiment used the purified enzyme Cyc1 from step 2 of example 2, which was purified after induction of strain expression at 30 ℃.
1. Influence of temperature on the Activity of the pure enzyme of the hexavalent chromium reductase Cyc1
The pure enzyme of the hexavalent chromium reductase Cyc1 is treated for 30min at the temperature of 25, 27, 30, 33, 35, 37, 40, 42 and 45 ℃ respectively, and a sample is taken to detect the activity of the hexavalent chromium reductase.
The detection method comprises the following steps: in 20mM MOPS-NaOH buffer containing 100mM NAD (P) H, 10mM FMN, 1mM CrO 3 And 10mM CaCl 2 50 μ L of Cyc1 pure enzyme was added, the reaction was carried out for 1min under the condition of pH =8.5, and the Cr (VI) content was measured by the dibenzoyl dihydrazide method.
As shown in FIG. 5, the activity of pure enzyme of the hexavalent chromium reductase Cyc1 was the maximum at 30 ℃ and the activity was relatively high at 27 ℃ and 33 ℃, which were 83% and 84% of the maximum activity, respectively; the activity at 25 ℃ and 42 ℃ is 40% and 32% of the maximum activity; it is thus clear that hexavalent chromium reductases are active over a wide temperature range.
2. Effect of pH on the Activity of the pure enzyme of the hexavalent chromium reductase Cyc1
The activity of the hexavalent chromium reductase is detected under the conditions of different pH values (6, 7, 8, 8.5, 9, 10, 11 and 12).
The detection method comprises the following steps: in 20mM MOPS-NaOH buffer containing 100mM NAD (P) H, 10mM FMN, 1mM CrO 3 And 10mM CaCl 2 Adding 50 mu L of Cyc1 pure enzyme, reacting for 1min at 30 ℃, and detecting the content of Cr (VI) by a diphenylcarbodihydrazide method.
As shown in FIG. 6, the hexavalent chromium reductase had the maximum activity at pH 8.5, and had relatively high activities at pH 8 and 9, which were 60% and 91%, respectively, of the maximum activity. The activity was 10% and 5% of the maximum activity at pH 6 and 10. Thus, hexavalent chromium reductase is relatively specific to the PH requirement.
3. Influence of metal ions on activity of pure enzyme of hexavalent chromium reductase Cyc1
Different metal ion (Na) + 、K + 、Mg 2+ 、Mn 2+ 、Zn 2+ 、Ca 2+ 、Cu 2+ EDTA) was detected under the conditions of the enzyme activity of the pure enzyme of the hexavalent chromium reductase Cyc1.
The specific method comprises the following steps: 10mM of different metal ions (10 mM in concentration) in 20mM MOPS-NaOH buffer containing 100mM NAD (P) H, 10mM FMN and 1mM CrO 3 50 μ L of Cyc1 pure enzyme was added, and the mixture was reacted at 30 ℃ and pH =8.5 for 1min, and the Cr (VI) content was measured by the diphenylcarbodihydrazide method.
As a result, as shown in FIG. 7, the enzyme purified from the hexavalent chromium reductase Cyc1 was Ca-containing 2+ Having maximum activity under Mg 2+ The higher activity is 60% of the maximum activity. Therefore, the hexavalent chromium reductase Cyc1 is relatively exclusively required for ions.
4. Effect of different concentrations of Cr (VI) on the Activity of hexavalent chromium reductase
The activity of the pure enzyme of the hexavalent chromium reductase Cyc1 is detected under the condition of different Cr (VI) concentrations.
The specific method comprises the following steps: in 20mM MOPS-NaOH buffer containing 100mM NAD (P) H, 10mM FMN and 50. Mu.L Cyc1 pure enzyme, 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM CrO was added 3 The system is reacted for 1min at the temperature of 30 ℃ and the pH =8.5, and the content of Cr (VI) is detected by a dibenzoyl dihydrazide method.
As shown in FIG. 8, the pure enzyme of the hexavalent chromium reductase Cyc1 had the maximum activity at a Cr (VI) concentration >0.4mM and a Km value of 0.2mM.
5. Degradation of Cr (VI) by recombinant strain
The fermentation broth of the genetically engineered bacteria E.coli BL21 (DE 3)/pXMJ-19-Cyc 1 and E.coli BL21 (DE 3)/pXMJ-19 prepared in example 2 were inoculated to the control strain to which 1.0mM CrO had been added in an inoculum size of 1% (v/v) 3 50mL of LB liquid medium (Amersham pharmacia Biotech) was cultured at 30 ℃ and 180rpm for 24 hours, and the Cr (VI) content was measured every 4 hours by the dibenzoyl dihydrazide method, and the results are shown in FIG. 9.
Results show that the capability of the strain for degrading Cr (VI) is obviously enhanced after the genetic engineering strain E.coli BL21 (DE 3) introduces exogenous Cyc1 gene.
Example 4: application of hexavalent chromium reductase Cyc1 pure enzyme in treatment of Cr (VI) containing wastewater
The method comprises the following specific steps:
(1) Taking a certain amount of Cr (VI) -containing wastewater from a tin-free wastewater field, carrying out high-sensitivity quantitative detection on Cr (VI) by using a dibenzoyl dihydrazide method, carrying out physical and chemical property detection on the wastewater, and determining that the wastewater contains Cr (VI) and the concentration of hexavalent chromium Cr (VI) in the wastewater is 100mg/L.
(2) The purified enzyme of the hexavalent chromium reductase Cyc1 prepared in example 2 (purified enzyme of Cyc1 obtained by inducing the expression of the strain at 30 ℃ in step 2) was added in an amount of 0.1U/mg Cr (VI) (i.e., 1U), treated at 30 ℃ and pH 8.5 for 6 hours, and the concentration of residual Cr (VI) was measured, and a control group without hexavalent chromium reductase was set, and the results are shown in FIG. 10.
As can be seen from FIG. 10, the Cr (VI) concentration in the wastewater without the addition of the hexavalent chromium reductase did not change significantly, and the Cr (VI) concentration in the wastewater with the addition of the hexavalent chromium reductase was reduced to 62mg/L, and the reduction rate was 38%, so that the hexavalent chromium in the wastewater could be removed rapidly and effectively.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> old yellow enzyme OYE2 protein and application thereof in chromium pollution
<130> BAA211189A
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 360
<212> PRT
<213> Artificial sequence
<400> 1
Val Ser Lys Leu Phe Thr Pro Ile Gln Ile Arg Asp Ile Thr Ile Pro
1 5 10 15
Asn Arg Val Trp Met Ser Pro Met Cys Thr Tyr Ser Ala Ala Thr Gly
20 25 30
Leu Gly Leu Pro Thr Asp Phe His Gln Ala His Tyr Ala Ala Arg Ala
35 40 45
Ala Gly Gly Val Gly Leu Val Met Val Glu Ala Thr Gly Val Ser Pro
50 55 60
Val Ala Pro Ile Ser Pro Val Asp Leu Gly Leu Trp Ser His Asp Gln
65 70 75 80
Ile Glu Pro Phe Ser Arg Val Thr Ala Ala Ile Arg Ala Gly Gly Ala
85 90 95
Val Pro Ala Val Gln Leu Ala His Ala Gly Arg Lys Ala Ser Thr Asp
100 105 110
Ala Pro Trp Asn Gly Gly Gly Tyr Val Gly Pro Glu Thr Asn Gly Trp
115 120 125
Glu Thr Val Gly Pro Ser Pro Leu Ala Phe Pro Gly Leu Pro Ala Pro
130 135 140
Arg Glu Leu Thr Val Ser Glu Ile Gln Glu Val Val Gln Gln Phe Ala
145 150 155 160
Gly Ala Ala Val Arg Ala Asp Gln Ala Gly Phe Asp Val Val Glu Ile
165 170 175
His Ala Ala His Gly Tyr Leu Leu His Asn Phe Leu Ser Pro Ile Ser
180 185 190
Asn Lys Arg Thr Asp Ser Tyr Gly Gly Ser Leu Glu Asn Arg Ala Arg
195 200 205
Ile Val Leu Glu Val Ile Asp Ala Ile Arg Ala Val Trp Pro Glu Glu
210 215 220
Lys Pro Val Phe Met Arg Ile Ser Thr Thr Asp Trp Val Glu Glu Asn
225 230 235 240
Pro Gln Asp Asp Arg Glu Ser Trp Thr Leu Ser Gln Ser Arg Gln Leu
245 250 255
Ala Leu Trp Ala Ser Glu Arg Gly Ile Asp Leu Ile Asp Ala Ser Ser
260 265 270
Gly Gly Leu Asp Ile Val Pro Ile Pro His Asp Arg Asp Tyr Gln Thr
275 280 285
Ala Lys Ala Ala Asp Leu His Ala Ser Thr Gly Ala Thr Val Ala Ala
290 295 300
Val Gly Arg Ile Asp Asp Ala Gln Thr Ala His Asn Leu Val Asp Ser
305 310 315 320
Gly Asp Val Asn Ala Val Phe Leu Gly Arg Pro Leu Leu Lys Asp Pro
325 330 335
Ser Trp Ala Asn Gln Ala Ala Leu Thr Leu Gly Ala Glu Pro Arg Tyr
340 345 350
Val Arg Gln Tyr Asp Tyr Val Leu
355 360
<210> 2
<211> 1083
<212> DNA
<213> Artificial sequence
<400> 2
gtgtccaagc tgtttacccc aattcaaatc cgcgacatca ccatccccaa ccgcgtgtgg 60
atgtcaccga tgtgcaccta ctctgcagcc accggtttag gtcttcccac cgattttcac 120
caggctcatt acgcagctcg cgcagcaggt ggtgtcggat tagtcatggt tgaagcaact 180
ggagtgagcc ccgtagctcc catctcccca gtcgaccttg gactttggag ccatgaccaa 240
attgaaccat tctcccgagt gacagcagct attcgcgccg gtggggcagt accggccgtt 300
caattagccc atgctggccg caaggcatcc accgatgctc cgtggaatgg tggcggatat 360
gttggaccag aaaccaatgg atgggagact gtcggcccca gccctctggc attcccaggt 420
ttgcctgctc cgcgcgagct gacggtttca gaaatccaag aggttgtgca gcagttcgct 480
ggcgccgccg ttcgtgccga tcaggctggt tttgatgtcg tggaaattca cgcagcacac 540
ggctaccttt tgcataactt cctttctccg atctccaaca agcgcaccga ttcatacggc 600
ggatctttag aaaaccgcgc tcgcatcgtg ctcgaagtca ttgatgcaat ccgcgcagtg 660
tggccagagg aaaagcctgt attcatgcgc atttccacca ccgactgggt agaggaaaac 720
ccacaggatg atcgcgagtc ctggacgctg agccaaagca ggcagctggc tttgtgggca 780
tccgagcgcg gaattgattt gatcgatgcc tcttctggtg gcctcgacat cgtccccatt 840
ccgcatgacc gcgattacca aaccgcgaag gccgcagatc ttcacgcaag taccggagcg 900
acagtcgctg ctgtggggcg cattgatgac gcccaaactg cgcacaattt ggttgattct 960
ggcgatgtta atgcagtttt cctcggccgt ccactgctca aggatccttc ctgggcaaac 1020
caagcagccc tcacgctagg tgcggaaccc aggtatgttc gccaatacga ctacgtactt 1080
taa 1083
Claims (8)
1. A method for degrading hexavalent chromium by an enzyme method is characterized in that hexavalent chromium reductase with an amino acid sequence shown as SEQ ID NO.1 is added into a reaction system containing hexavalent chromium for degradation.
2. The method of claim 1 wherein the nucleotide sequence encoding said hexavalent chromium reductase is set forth in SEQ ID No. 2.
3. The process of claim 1 or 2 wherein the hexavalent chromium reductase is added in an amount of at least 0.1U/mg hexavalent chromium Cr (VI) in the reaction system.
4. A method for degrading hexavalent chromium through whole cells is characterized in that microbial cells containing hexavalent chromium reductase of which the amino acid sequence is shown as SEQ ID NO.1 are added into a reaction system containing hexavalent chromium for degradation.
5. The method of claim 4 wherein the nucleotide sequence encoding said hexavalent chromium reductase is set forth in SEQ ID No. 2.
6. The method of claim 5, wherein the microbial cell is a recombinant E.coli, and the recombinant E.coli is a recombinant E.coliE.coliBL21 (DE 3) is used as an expression host, and pXMJ-19 is used as an expression vector.
7. The method of claim 6, wherein the recombinant E.coli is added to the reaction system in an amount of at least 1% by volume.
8. A method for improving the degradation capability of Escherichia coli on hexavalent chromium is characterized in that the method is to express hexavalent chromium reductase of which the amino acid sequence is shown as SEQ ID No.1 in the Escherichia coli.
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