CN113735282B - Old yellow enzyme OYE2 protein and application thereof in chromium pollution - Google Patents

Abstract

The invention discloses an old yellow enzyme OYE2 protein and its application in chromium pollution, belonging to the field of heavy metal prevention and control. The present invention uses the old yellow enzyme OYE2 protein in Corynebacterium crenatum SYPA 5‑5 as an active ingredient for reducing Cr(VI), which has the activity of hexavalent chromium reductase, and the chromium reductase has a strong reduction effect on Cr(VI). It can quickly remove Cr(VI) in wastewater. Adopt method of the present invention, in the waste water that does not add hexavalent chromium reductase of the present invention, Cr (VI) concentration does not change significantly, adds Cr (VI) concentration in the waste water of hexavalent chromium reductase of the present invention and drops to 62mg/L, with a reduction rate of 38%, it can quickly and effectively remove hexavalent chromium in wastewater. Therefore, using one or more of these enzymes to prepare hexavalent chromium reductase to treat Cr(VI)-containing wastewater has potential for industrial application.

Description

An Old Yellow Enzyme OYE2 Protein and Its Application in Chromium Pollution

technical field

The invention relates to an old yellow enzyme OYE2 protein and its application in chromium pollution, belonging to the field of heavy metal prevention and control.

Background technique

Chromium (Cr) pollution in soil and groundwater is one of the most concerned environmental problems in the world, and it mainly comes from industries such as chromium salt production, electroplating, leather tanning, pigment production, mining and metallurgy. Chromium exists in two stable chromium oxidation states in the environment: hexavalent chromium and trivalent chromium; compared with hexavalent chromium Cr(VI), trivalent chromium Cr(III) has low solubility, weak migration ability and low biological toxicity . Therefore, one of the effective strategies for the treatment of chromium pollution in water is to reduce hexavalent chromium Cr(VI) to trivalent chromium Cr(III). Among them, microbial reduction of hexavalent chromium Cr(VI) is regarded as a method with great application potential, but the microbial remediation process still has the disadvantage of a long cycle, and the use of microorganisms for in-situ remediation is greatly affected by environmental factors. There is mutual competition with indigenous microorganisms, resulting in unstable restoration effect.

At present, there are a large number of research reports on the use of hexavalent chromium reductase to repair soil and control polluted groundwater, such as using laccase to repair DDT-contaminated soil, soil enzymes to repair lead-contaminated soil, immobilized enzymes to repair soil contaminated by pesticides, and nitrifying bacteria. Enzyme denitrification treatment of groundwater, etc. For Cr(VI) contamination, a large number of studies have reported Cr(VI) reductases, such as NfsA and YieF enzymes from Escherichia coli, whose maximum reduction rates are 0.25 and 5.0 μmol/min/mg, respectively; Pseudomonas putida KT2400 The maximum reduction rate of ChrR is 8.8μmol/min/mg. All these proteases can effectively reduce Cr(VI) under certain conditions, but there is still a big gap from large-scale industrial application. Therefore, there is an urgent need for a method for efficiently controlling chromium pollution in water to meet large-scale industrial applications.

Contents of the invention

In order to obtain a method for efficiently treating chromium pollution in water bodies, the invention provides a hexavalent chromium reductase and its application in treating chromium pollution in water bodies. The application refers to putting the hexavalent chromium reductase into the hexavalent chromium polluted water body to reduce the highly water-soluble hexavalent chromium to the low water-soluble trivalent chromium, and achieve the purpose of treatment by reducing its mobility.

The invention provides a method for enzymatically degrading hexavalent chromium. The method includes adding hexavalent chromium reductase with the amino acid sequence shown in SEQ ID NO.1 to a reaction system containing hexavalent chromium for degradation.

In one embodiment of the present invention, the nucleotide sequence encoding the hexavalent chromium reductase is shown in SEQ ID NO.2. Its annotated result in GenBank is riboflavin oxidoreductase/NADH oxidase, and this protein belongs to the OYE2 family member, which is named Cyc1 in the present invention.

In one embodiment of the present invention, the hexavalent chromium reductase is derived from Corynebacterium crenatum SYPA5-5.

In one embodiment of the present invention, in the reaction system, the added amount of the hexavalent chromium reductase is at least 0.1 U/mg hexavalent chromium Cr(VI).

In one embodiment of the present invention, the dosage of the hexavalent chromium reductase is at least 0.1 U/mg hexavalent chromium Cr(VI).

In one embodiment of the present invention, when the chromium concentration in the polluted water body is ≤100 mg/L, the dose of hexavalent chromium reductase is 0.1 U/mg Cr(VI). Calculated according to the kinetic maximum velocity Vmax=30.39umol Cr(VI)/min/mg enzyme protein.

In one embodiment of the present invention, the degradation reaction condition is: carried out under the condition of pH 6-12.

In one embodiment of the present invention, the optimal pH value of the enzyme is 8.5.

In one embodiment of the present invention, the degradation reaction is carried out at a temperature of 25-40°C.

In one embodiment of the present invention, the optimum action temperature of the hexavalent chromium reductase is 30°C.

The present invention also provides a recombinant Escherichia coli containing the hexavalent chromium reductase whose amino acid sequence is shown in SEQ ID NO.1.

In one embodiment of the present invention, the nucleotide sequence encoding the hexavalent chromium reductase is shown in SEQ ID NO.2.

In one embodiment of the present invention, the recombinant Escherichia coli uses E. coli BL21(DE3) as the expression host and pXMJ-19 as the expression vector.

The present invention also provides a method for degrading hexavalent chromium by whole cells, the method is: adding the microbial cell containing the hexavalent chromium reductase as shown in SEQ ID NO.1 to the reaction containing hexavalent chromium degradation in the system.

In one embodiment of the present invention, the reaction system is a sewage environment.

In one embodiment of the present invention, the nucleotide sequence encoding the hexavalent chromium reductase is shown in SEQ ID NO.2.

In one embodiment of the present invention, the microbial cell is recombinant Escherichia coli.

In one embodiment of the present invention, the recombinant Escherichia coli uses E. coli BL21(DE3) as the expression host and pXMJ-19 as the expression vector.

In one embodiment of the present invention, the amount of the recombinant Escherichia coli added to the reaction system is at least 1% (v/v).

The present invention also provides a method for improving the degradation ability of Escherichia coli to hexavalent chromium, the method is, expressing the hexavalent chromium reductase in Escherichia coli whose amino acid sequence is shown as SEQ ID NO.1.

In one embodiment of the present invention, the nucleotide sequence encoding the hexavalent chromium reductase is shown in SEQ ID NO.2.

The present invention also provides a hexavalent chromium reductase with an amino acid sequence as shown in SEQ ID NO.1, or a recombinant vector containing a hexavalent chromium reductase with a nucleotide sequence as shown in SEQ ID NO.2, or with an amino acid sequence Application of the microbial cell of hexavalent chromium reductase shown in SEQ ID NO.1 in the preparation of products degrading hexavalent chromium.

In one embodiment of the present invention, the recombinant vectors use pET-28a, pDXW-10, pXMJ-19 as expression vectors.

In one embodiment of the present invention, the microbial cells use bacteria or fungi as host cells.

In one embodiment of the present 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 present invention also provides a preparation method of hexavalent chromium reductase that can quickly remove Cr(VI) in wastewater, construct the above-mentioned hexavalent chromium reductase gene Cyc1 on the expression vector, and use the prokaryotic expression system to prepare highly active chromium Reductase Cyc1.

In one embodiment of the present invention, the Corynebacterium crenatum SYPA 5-5 genome is used as a template, the following primers are designed, and the nucleic acid sequence (as shown in SEQ ID NO.2) of the chromium reductase gene Cyc1 is obtained by PCR amplification. The nucleic acid sequence is constructed on the pXMJ-19 vector, and transformed into BL21 Escherichia coli competent cells, the obtained recombinant bacteria are induced to express, and the target protein is obtained by separation and purification. The primer sequences involved are as follows:

F: 5'-AGCTTAAAGGAGGACAACCGTGCACCACCACCACCACCACCTCCAAGCTGT-3'

R: 5'-AGCCAAGTGAATTCTTAAAAGTACGTAGTC-3'.

Beneficial effect

(1) The chromium reductase derived from Corynebacterium crenatum SYPA5-5 provided by the present invention has a strong reducing effect on Cr(VI), and can quickly remove Cr(VI) in wastewater.

(2) Adopt the method of the present invention, in the waste water that does not add hexavalent chromium reductase of the present invention, Cr (VI) concentration does not change obviously, add Cr (VI) in the waste water of hexavalent chromium reductase of the present invention The concentration drops to 62mg/L, and the reduction rate reaches 38%, which can quickly and effectively remove hexavalent chromium in wastewater. Therefore, using one or more of these enzymes to prepare hexavalent chromium reductase to treat Cr(VI)-containing wastewater has potential for industrial application.

Description of drawings

Figure 1: Electrophoretic pattern of PCR amplification results of Cr(VI) reduced gene Cyc1 in Example 1 of the present invention; wherein, M: nucleic acid marker; lanes 1, 2, 3 and 4: PCR amplification products of Cyc1.

Figure 2: Electropherogram of the result of double digestion of expression vector pXMJ-19 in Example 1 of the present invention; wherein, M: nucleic acid marker; lanes 1 and 2: double digestion of pXMJ-19 (EcoR I and Hind III).

Figure 3: SDS-PAGE image of the crude enzyme solution of chromium reductase Cyc1 in Example 2 of the present invention; wherein, M: protein marker; lane 1: pXMJ19 empty control; lane 2: Cyc1 protein expression induced at 15°C; lane 3: Cyc2 protein expression was induced at 20°C; Swimming lane 4: Cyc1 protein expression was induced at 25°C; Swimming lane 5: Cyc1 protein expression was induced at 30°C.

Figure 4: SDS-PAGE image of purified chromium reductase Cyc1 in Example 2 of the present invention; among them, M: protein marker; lane 1: Cyc1 protein expression induced at 30°C; lane 2: Cyc2 protein expression induced at 25°C; lane 3 : Cyc1 protein expression was induced at 20°C.

Figure 5: Effect of temperature on the activity of hexavalent chromium reductase in Example 3 of the present invention.

Fig. 6: Effect of pH value on activity of hexavalent chromium reductase in Example 3 of the present invention.

Figure 7: Effects of different metal ions on the activity of hexavalent chromium reductase in Example 3 of the present invention.

Fig. 8: Effects of different concentrations of Cr(VI) on the activity of hexavalent chromium reductase in Example 3 of the present invention.

Figure 9: Degradation of Cr(VI) by recombinant bacteria in Example 3 of the present invention.

Figure 10: Reduction of Cr(VI)-containing wastewater by hexavalent chromium reductase in Example 4 of the present invention.

Detailed ways

Corynebacterium crenatum SYPA 5-5 is a strain of L-arginine high-yielding bacteria selected by our laboratory. Corynebacterium crenatum SYPA5-5 is recorded in the patent application text with publication number CN1441055A, and the preservation number is CGMCC NO.0890 (the strain number of this strain in the patent application text is SDNN403, and the inventor renumbered it as SYPA5-5 during the experiment); based on the analysis of the previous transcriptome and proteome data, it was found that hexavalent chromium reductase ( Cyc1) was up-regulated under high dissolved oxygen relative to low dissolved oxygen conditions. In order to further study the anti-oxidative stress function of Cyc1, a Cyc1 overexpression strain and a knockout strain were constructed, and it was found that the Cyc1 protein has extremely strong redox properties. Taking Cyc1 as the research object, the circular dichroism structure of Cyc1 was analyzed, and the SWISS-MODEL modeling analysis was used to detect its ability to reduce Cr(VI), and the structure and function of Cyc1 were verified. The Cyc1 protein has a strong reducing effect on Cr(VI), can quickly remove Cr(VI) in wastewater, and has important application value in the treatment of Cr(VI)-containing wastewater.

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

The medium involved in the following examples is as follows:

LB liquid medium (g/L): 5 yeast powder, 10 tryptone, 10 sodium chloride.

LB solid medium: on the basis of LB liquid medium, add 1.5-2.0% agar powder.

The detection methods involved in the following examples are as follows:

Detection of protein concentration:

The protein concentration was determined using a Bradford kit, with bovine serum albumin as a standard.

Detection of Cr(VI) concentration:

Diphenylcarbazide method, extract 0.3ml of the sample to be tested, add 2.5ml 0.12M concentrated sulfuric acid solution, add a volume of 0.2ml diphenylcarbazide (dissolved in acetone) to the above mixture, and make its final concentration reach 0.4mM, detected at 543nm absorbance.

The extraction method of genomic DNA involved in the following examples and related plasmids:

Corynebacterium crenatum SYPA5-5 was used to extract genomic DNA according to the instructions of the Shanghai Jierui Bacteria Genomic DNA Rapid Extraction Kit. When extracting the plasmid, centrifuge the cultured bacterial solution with an appropriate concentration and remove the supernatant. Corynebacterium glutamicum is a Gram-positive bacterium. When extracting the plasmid from its recombinant strain, it is necessary to add an appropriate amount of lysozyme and put it in a water bath at 37°C for 0.5h. There is no need to add lysozyme when extracting the plasmid from the recombinant strain of Bacillus, and it is extracted with the Jierui Small Plasmid Extraction Kit, and the detailed operation refers to the instruction manual.

The Escherichia coli competent preparation and chemical transformation method involved in the following examples are as follows:

To prepare Escherichia coli transfected competent cells, the Competent Cell Preparation Kit from TakaRa Company was used, and the detailed operation was referred to the instruction manual. Transformed into E.coliBL21 by heat shock at 42°C, positive transformants were obtained through antibiotic resistance plate screening, the plasmid was extracted for PCR verification and sent to Jinweizhi Biotechnology Company for sequencing verification.

Embodiment 1: Construction can express the genetically engineered bacterium of Cr (VI) reductase gene Cyc1

1. Cloning of Cyc1 gene

The target gene (the nucleotide sequence is shown in SEQ ID NO.2) is amplified by PCR using the high-fidelity enzyme PhantaR Max (p515) DNA polymerases. The PCR system is a 50 μL standard reaction system. The PCR amplification program is: 95°C Pre-denaturation for 5 minutes; denaturation at 95°C for 30s; annealing temperature is generally set at 58-60°C, 30-60s; extension at 72°C is set according to the amplification of 1500bp genes per minute; denaturation to extension program is 30 cycles; 72°C is extended for 5 minutes; Store at 4°C. After PCR, the PCR products were subjected to nucleic acid electrophoresis for verification and gel recovery and purification. The results are shown in Figure 1. After recovery, double enzyme digestion was performed with the linearized vector expression vector pXMJ-19, the result is shown in Figure 2; after connection with ClonExpress II One Step Cloning Kit, the E.coli BL21 (DE3) competent cells were transformed, and the positive transformants were picked and extracted Plasmids were verified by PCR and sequencing.

The genetically engineered strain E.coli BL21(DE3)/pXMJ-19-Cyc1 expressing hexavalent chromium reductase Cyc1 was prepared.

The empty pXMJ-19 plasmid was introduced into Escherichia coli E. coli BL21 (DE3) competent cells according to the above method, and the control strain E. coli BL21 (DE3)/pXMJ-19 was prepared.

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(DE3)/pXMJ-19-Cyc1 and E.coliBL21(DE3)/pXMJ-19 control strains prepared in Example 1 were respectively streaked onto the LB solid medium containing Chl ^R , After activation at 37°C and 12h, single colonies were picked and inserted into 10mL vials of LB liquid medium containing Chl ^R , cultured at 30°C and 180rpm for 16-24h, and seed solutions were prepared respectively;

The prepared seed solution was transferred to 4 bottles of 50mL LB liquid medium containing Chl ^R according to the inoculation amount of 1% (v/v). After culturing at 30°C and 180rpm for 4-5h, isopropyl β- D-1-thiogalactoside (IPTG) to a final concentration of 1mmol·L ^-1 , induced expression at 15°C, 20°C, 25°C, 30°C, 180rpm for 16-18h, prepared at different temperatures induced fermentation broth;

The prepared fermentation broth was centrifuged at 4° C. and 8000 rpm for 10 min to collect bacterial cells.

The collected cells were washed twice with 0.1 mol·L ^- 1 PBS buffer (pH 7.4), and then resuspended in PBS buffer (pH 7.4) to control the cell concentration (cell concentration: OD ₆₀₀ = 6.0) In the case of consistency, use an ultrasonic breaker to crush, centrifuge the crushed solution at 4°C for 20 minutes, collect the supernatant, and analyze the protein expression by SDS-PAGE. The results are shown in Figure 3. From Figure 3, it can be seen that hexavalent chromium reductase Cyc1 protein expression was best induced at 30°C; the remaining crude enzyme solution was used for subsequent enzyme activity assays.

The crude enzyme solution containing the hexavalent chromium reductase Cyc1 induced and expressed at different temperatures was prepared.

2. Purification of hexavalent chromium reductase Cyc1 protein

The crude enzyme solution containing hexavalent chromium reductase Cyc1 prepared in step 1 was filtered with a 0.45 μm filter membrane, and then purified with a Ni-NTA protein purification column to remove impurities. The Ni ⁺ on the column is chelated, and the gradient elution is carried out, and the gradient elution of the imidazole buffer with different concentrations is used. The purified protein bound to the purification column is eluted, and then the purified enzyme is obtained. Then the Cyc1 pure enzyme solution was analyzed by SDS-PAGE, as shown in Figure 4. From Figure 4, it can be seen that the expression effect of the hexavalent chromium reductase Cyc1 protein is best induced at 30°C.

The concentration of hexavalent chromium reductase Cyc1 protein under induction expression at 30°C was determined using Bradford kit, with bovine serum albumin as the standard.

The specific enzyme activity data of hexavalent chromium reductase Cyc1 pure enzyme was contained in 20mM MOPS-NaOH buffer, which contained 100mM NAD(P)H, 10mM FMN, 1mM CrO ₃ and 10mM CaCl ₂ , added 50μL Cyc1 pure enzyme, in The reaction was carried out at 30° C. and pH=8.5 for 1 min, and the Cr(VI) content was detected by the diphenylcarbazide method. The results are shown in Table 1.

Table 1 Specific enzyme activity of Cyc1 pure enzyme solution for Cr(VI) reduction under different ligands (U/mg)

Embodiment 3: Activity analysis of pure enzyme of hexavalent chromium reductase Cyc1

The following experiments used the purified Cyc1 enzyme obtained in step 2 of Example 2 after the expression of the induced strain at 30°C.

1. Effect of temperature on the activity of pure enzyme of hexavalent chromium reductase Cyc1

The pure enzyme of hexavalent chromium reductase Cyc1 was treated at 25, 27, 30, 33, 35, 37, 40, 42 and 45°C for 30 minutes, and samples were taken to detect the activity of hexavalent chromium reductase.

The detection method is: in 20mM MOPS-NaOH buffer, the buffer contains 100mM NAD(P)H, 10mM FMN, 1mM CrO ₃ and 10mM CaCl ₂ , add 50μL of Cyc1 pure enzyme, and react for 1min under the condition of pH=8.5 , Cr(VI) content was detected by the diphenylcarbazide method.

The results are shown in Figure 5, the pure enzyme of hexavalent chromium reductase Cyc1 has the maximum activity at 30°C, and has relatively high activity at 27°C and 33°C, which are 83% and 84% of the maximum activity respectively; At 42°C, its activity is 40% and 32% of the maximum activity; thus, it can be seen that the hexavalent chromium reductase has activity in a wide temperature range.

2. The effect of pH on the activity of pure enzyme of hexavalent chromium reductase Cyc1

The activity of hexavalent chromium reductase was detected under different pH values (6, 7, 8, 8.5, 9, 10, 11, 12).

The detection method is: in 20mM MOPS-NaOH buffer, the buffer contains 100mM NAD(P)H, 10mM FMN, 1mM CrO ₃ and 10mM CaCl ₂ , add 50μL Cyc1 pure enzyme, react at 30°C for 1min, Cr(VI) content was detected by the diphenylcarbazide method.

The results are shown in Figure 6, the hexavalent chromium reductase has the maximum activity at pH 8.5, and has relatively high activity at pH 8 and 9, respectively 60% and 91% of the maximum activity. When the pH is 6 and 10, its activity is 10% and 5% of the maximum activity. It can be seen that hexavalent chromium reductase is relatively specific to the pH requirement.

3. Effect of metal ions on the activity of pure enzyme of hexavalent chromium reductase Cyc1

The activity of pure enzyme of hexavalent chromium reductase Cyc1 was detected under different metal ions (Na ⁺ , K ⁺ , Mg ²⁺ , Mn ²⁺ , Zn ²⁺ , Ca ²⁺ , Cu ²⁺ , EDTA).

The specific method is as follows: 10 mM different metal ions (concentration: 10 mM) in 20 mM MOPS-NaOH buffer, the buffer contains 100 mM NAD(P)H, 10 mM FMN and 1 mM CrO ₃ , add 50 μL Cyc1 pure enzyme, in React for 1 min under the conditions of 30°C and PH=8.5, and detect the Cr(VI) content by the diphenylcarbazide method.

The results are shown in FIG. 7 , the pure enzyme of hexavalent chromium reductase Cyc1 has the maximum activity under the condition of Ca ²⁺ , and has a relatively high activity under the condition of Mg ²⁺ , which is 60% of the maximum activity. It can be seen that the pure enzyme of hexavalent chromium reductase Cyc1 has relatively specific requirements for ions.

4. The effect of different concentrations of Cr(VI) on the activity of hexavalent chromium reductase

The activity of the pure enzyme of hexavalent chromium reductase Cyc1 was detected under different Cr(VI) concentration conditions.

The specific method is as follows: In 20mM MOPS-NaOH buffer, which contains 100mM NAD(P)H, 10mM FMN and 50μL Cyc1 pure enzyme, add 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM , 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM CrO ₃ , the system was reacted at 30°C and pH=8.5 for 1min, and the Cr(VI) content was detected by the diphenylcarbazide method.

The results are shown in FIG. 8 , the pure enzyme of hexavalent chromium reductase Cyc1 has the maximum activity when the concentration of Cr(VI)>0.4mM, and the Km value is 0.2mM.

5. Degradation of Cr(VI) by recombinant strains

Example 2 is prepared to obtain the fermented liquid of genetically engineered bacteria E.coli BL21 (DE3)/pXMJ-19-Cyc1, E.coli BL21 (DE3)/pXMJ-19 control strain, inoculated by 1% (v/v) The amount was transferred to 50mL LB liquid medium that had been added with 1.0mM CrO ₃ , cultured at 30°C and 180rpm for 24 hours, and the content of Cr(VI) was detected every 4 hours by the diphenylcarbazide method. As shown in Figure 9.

The results showed that the ability of the strain to degrade Cr(VI) was significantly enhanced after the introduction of exogenous Cyc1 gene into the genetically engineered strain E.coli BL21(DE3).

Embodiment 4: the application of hexavalent chromium reductase Cyc1 pure enzyme in the treatment of Cr(VI)-containing wastewater

Specific steps are as follows:

(1) A certain amount of Cr(VI)-containing wastewater was taken from a wastewater field in Wuxi, and the high-sensitivity quantitative detection of Cr(VI) was carried out by the diphenylcarbazide method. The physical and chemical properties of the wastewater were tested, and the The waste water contains Cr(VI), and the concentration of hexavalent chromium Cr(VI) in the waste water is 100 mg/L.

(2) Add the hexavalent chromium reductase Cyc1 pure enzyme prepared in Example 2 (the purified Cyc1 purified enzyme expressed by the induced strain obtained in step 2 at 30°C) at the dosage of 0.1U/mg Cr(VI) (i.e. 1 U) was added therein, treated at 30°C and pH 8.5 for 6 hours, the residual Cr(VI) concentration was measured, and a control group without adding hexavalent chromium reductase was set up, the results are shown in Figure 10.

It can be seen from Figure 10 that in the wastewater without hexavalent chromium reductase, the concentration of Cr(VI) did not change significantly, and the concentration of Cr(VI) in the wastewater added with hexavalent chromium reductase dropped to 62mg/L, and the reduction rate reached 38%, can quickly and effectively remove hexavalent chromium in wastewater.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

SEQUENCE LISTING

<110> Jiangnan University

<120> An old yellow enzyme OYE2 protein and its application 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 enzymatically degrading hexavalent chromium, characterized in that a hexavalent chromium reductase with an amino acid sequence as shown in SEQ ID NO.1 is added to a reaction system containing hexavalent chromium for degradation. 2. The method according to claim 1, characterized in that the nucleotide sequence encoding the hexavalent chromium reductase is shown in SEQ ID NO.2. 3. The method according to claim 1 or 2, characterized in that, in the reaction system, the added amount of the hexavalent chromium reductase is at least 0.1 U/mg hexavalent chromium Cr(VI). 4. A method for degrading hexavalent chromium by whole cells, characterized in that, the method is that microbial cells containing amino acid sequences such as hexavalent chromium reductase shown in SEQID NO.1 are added to the reaction containing hexavalent chromium degradation in the system. 5. The method according to claim 4, characterized in that the nucleotide sequence encoding the hexavalent chromium reductase is shown in SEQ ID NO.2. 6 . The method according to claim 5 , wherein the microbial cell is a recombinant E. coli , and the recombinant E. coli uses E. coli BL21 (DE3) as an expression host and pXMJ-19 as an expression vector. 7. The method according to claim 6, wherein the amount of the recombinant Escherichia coli added to the reaction system is at least 1% by volume. 8. A method for improving the degradability of Escherichia coli to hexavalent chromium, characterized in that the method is to express a hexavalent chromium reductase with an amino acid sequence as shown in SEQ ID NO.1 in Escherichia coli.

Images