CN116200351B - Cr (VI) reductase, preparation method thereof and application thereof in chromium-polluted water body restoration - Google Patents
Cr (VI) reductase, preparation method thereof and application thereof in chromium-polluted water body restoration Download PDFInfo
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Abstract
The invention discloses a Cr (VI) reductase, a preparation method and application thereof in chromium-polluted water body restoration, wherein the amino acid sequence of the Cr (VI) reductase is shown as SEQ ID NO.1, the preparation method comprises the steps of connecting a fragment with a nucleotide sequence shown as SEQ ID NO.3 into a plasmid to obtain a recombinant plasmid, then converting the recombinant plasmid into escherichia coli BL21 (DE 3) to obtain recombinant bacteria, and collecting cells after the expression is finished, crushing and purifying the cells to obtain the recombinant bacteria. After the Cr (VI) reductase is added into a Cr (VI) polluted water body, the Cr (VI) with high biotoxicity and high water solubility can be quickly reduced into Cr (III) with low biotoxicity and low water solubility, so that a good repairing effect is achieved.
Description
Technical Field
The invention belongs to the technical field of heavy metal pollution control, and particularly relates to Cr (VI) reductase, a preparation method thereof and application thereof in chromium-polluted water body restoration.
Background
Heavy metal chromium (Cr) has strong biotoxicity and carcinogenicity to human and animal bodies, so chromium pollution in soil and water is an environmental problem of great concern. Chromium has corrosion resistance and high hardness, so that the chromium is widely applied to the industries of surface electroplating, textile dyeing, leather processing, electroplating surface treatment and the like. Current uncontrolled exploitation of chromium minerals and unordered discharge of chromium-containing industrial wastewater are important factors leading to chromium pollution in soil and water bodies. In view of the problems of heavy metal chromium pollution which are always present and severe in situation, an efficient and environment-friendly method for treating and repairing chromium pollution in the environment is a current urgent need.
Chromium in the environment mainly has two stable valence states of hexavalent chromium Cr (VI) and trivalent chromium Cr (III), wherein the biotoxicity of the Cr (VI) is far higher than that of the Cr (III), and the water solubility and migration capacity of the Cr (VI) are far higher than those of the Cr (III), so that one of the effective strategies for treating the chromium pollution in the water body is to reduce the Cr (VI) with high valence state into the Cr (III) with low valence state. At present, most of chromium pollution repair researches adopt a microbial reduction treatment method, and although the method has low cost efficiency and small influence on environment, the method has longer repair period, is greatly influenced by environmental biological and non-biological factors and has unstable repair effect.
Research shows that compared with the method of microbial reduction, the repairing method directly utilizing the microbial active enzyme preparation can greatly reduce the repairing period, has more stable repairing effect and has little secondary influence on the environment. The enzymes reported in the prior literature to be capable of reducing high-toxicity Cr (VI) to low-toxicity Cr (III) mainly comprise protein ChrR in Pseudomonas putida KT2400, wherein the maximum reduction rate is 8.8 mu mol min -1·mg-1, protein NfsA in ESCHERICHIA COLI and YIEF in ESCHERICHIA COLI, the maximum reduction rates are 0.25 and 5.0 mu mol min -1·mg-1 respectively, the maximum rate of reducing Cr (VI) by chromium reductase NirK-Cr of Pannonibacter phragmitetus BB is 34.46 mu mol min -1·mg-1, and the maximum rate of reducing Cr (VI) by old yellow enzyme OYE2 in Corynebacterium crenatum SYPA-5 is 30.39 mu mol min -1·mg-1. However, the reduction efficiency of the active enzyme preparation is still relatively low, and the industrial requirement cannot be met, so that the practical application of the active enzyme preparation restoration method is limited.
Disclosure of Invention
Therefore, the invention aims to provide the efficient protease with the Cr (VI) reduction function, which can quickly reduce Cr (VI) with high biotoxicity and high water solubility in Cr (VI) polluted water into Cr (III) with low biotoxicity and low water solubility, thereby achieving a good repair effect.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
The amino acid sequence of the Cr (VI) reductase provided by the invention is shown as SEQ ID NO.1, the Cr (VI) reductase is derived from Archaeoglobus fulgidus (Asgard), and the annotation result in the ArCOG database is nitroreductase nfnB.
The invention further provides a preparation method of the Cr (VI) reductase, which comprises the following steps:
s1, connecting a fragment with a nucleotide sequence shown as SEQ ID NO.3 into a plasmid to obtain a recombinant plasmid, and then converting the recombinant plasmid into escherichia coli BL21 (DE 3) to obtain recombinant bacteria, wherein the recombinant bacteria are induced to express by IPTG;
s2, after the expression is finished, centrifugally collecting cells, carrying out ultrasonic disruption after the cells are resuspended, centrifugally collecting supernatant, and purifying to obtain the pure enzyme of the Cr (VI) reductase.
In a specific embodiment of the present invention, the plasmid in step S1 is the expression vector pET-28a (+).
Further, when the plasmid is an expression vector pET-28a (+), the method for connecting the fragment with the nucleotide sequence shown as SEQ ID NO.3 to the plasmid is that an NcoI enzyme cutting site is added at the 5 'end of the fragment, an XhoI enzyme cutting site is added at the 3' end, and then the fragment and the expression vector pET-28a (+) are respectively connected after double enzyme cutting by using NcoI/XhoI.
In one embodiment of the present invention, the 3' end of the fragment in step S1 is linked to a histidine purification tag, so that the purification step in step S2 can be performed using His PurTM Ni-NTA purification kit.
The invention also provides application of the Cr (VI) reductase in repairing a Cr (VI) polluted water body, which comprises the steps of adding the Cr (VI) reductase into the Cr (VI) polluted water body, and reducing Cr (VI) with high biotoxicity and high water solubility into Cr (III) with low biotoxicity and low water solubility, thereby achieving the repairing effect.
The Cr (VI) reductase provided by the invention has Cr (VI) reducing activity in a wider temperature range of 20-50 ℃, but has a relatively specific pH requirement, has relatively high activity at pH 7 and 8, and almost loses activity at pH6 and 9. Therefore, the reduction conditions suitable for the Cr (VI) reductase are pH 7-8 and temperature 20-50 ℃.
Furthermore, in the technical scheme, the Cr (VI) polluted water body is also provided with the reduced coenzyme, and preferably the reduced coenzyme I disodium.
The invention has the beneficial effects that the nitroreductase nfnB in the archaea of Alasgard (Asgard) has a strong Cr (VI) reduction function, has good reduction activity in a wider temperature range, is suitable for repairing Cr (VI) water bodies in natural environments, has a high reduction removal rate of the nitroreductase nfnB, has a maximum reduction rate of 69.2 mu mol/min -1·mg-1 which is far higher than that of the prior reported enzyme, has important application value in Cr (VI) containing wastewater treatment, optimizes a coding sequence, and constructs a high-efficiency expression and purification method of the protease, thereby laying a foundation for further application.
Drawings
FIG. 1 is a map of recombinant plasmid nfnB-pET-28a (+) constructed in example 1 of the present invention;
FIG. 2 is a SDS-PAGE electrophoresis of the Cr (VI) reductase nfnB protein obtained by prokaryotic expression of E.coli in example 1 of the present invention before and after purification;
FIG. 3 is a graph showing the results of the detection of the Cr (VI) reducing activity of the Cr (VI) reductase nfnB protein of example 2 of the present invention;
FIG. 4 shows the effect of temperature on the activity of Cr (VI) reductase nfnB in example 3 of the present invention;
FIG. 5 shows the effect of pH on the activity of Cr (VI) reductase nfnB in example 4 of the present invention
FIG. 6 is a graph showing the results of the treatment of Cr (VI) containing wastewater with the Cr (VI) reductase nfnB protein of example 5 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples and experimental data in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In the examples which follow, the reagents and materials described are commercially available, unless otherwise indicated, according to the usual experimental conditions or according to the methods and conditions recommended in the product description.
Example 1
The amino acid sequence of the Cr (VI) reductase provided by the invention is shown as SEQ ID NO.1, is a protease from Archaeoglobus fulgidus BR.bin352, the nucleotide sequence for encoding the protease is shown as SEQ ID NO.2, and the annotation result in a ArCOG database is nitroreductase nfnB.
The example provides a preparation method of Cr (VI) reductase, which comprises the following steps:
(1) Codon optimization of the Cr (VI) reductase gene nfnB.
In order to increase the translation efficiency of a target gene in a bacterial host escherichia coli BL21 (DE 3), partial codons in a sequence shown in SEQ ID NO.2 are optimized according to the preference of codon usage of the escherichia coli so as to improve the expression quantity of the target gene nfnB in the escherichia coli BL21 (DE 3), and the nucleotide sequence after codon optimization is shown in SEQ ID NO. 3.
(2) An expression plasmid of Cr (VI) reductase nfnB was constructed.
After the Cr (VI) reductase gene nfnB is codon optimized, adding histidine purification tag at 3' end of nucleotide sequence, adding an NcoI enzyme cutting site at 5' end of nucleotide sequence, adding an XhoI enzyme cutting site at 3' end for subsequent cloning to expression vector, and subjecting the nucleotide sequence to total gene synthesis by Kirschner Biotechnology Co., ltd., china, nanjing.
The synthesized nfnB gene sequence and the expression vector pET-28a (+) plasmid are respectively subjected to double digestion by using NcoI and XhoI at 37 ℃, then are connected by using ClonExpress II one Step Cloning Kit and are transformed into E.coli DH5 alpha competent cells, positive transformant is selected to extract plasmid for sequencing verification, and finally recombinant plasmid nfnB-pET-28a (+) is obtained, and the structure diagram of the recombinant plasmid is specifically shown in figure 1.
(3) High-efficiency expression and purification of Cr (VI) reductase nfnB protein.
The recombinant plasmid nfnB-pET-28a (+) is transformed into escherichia coli BL21 (DE 3) to obtain recombinant bacteria BL21 (DE 3)/nfnB-pET-28 a (+) which is used for efficiently expressing Cr (VI) reductase nfnB protein, and the specific process is as follows:
Recombinant bacteria BL21 (DE 3)/nfnB-pET-28 a (+) was inoculated into LB medium containing 100. Mu.g/mL of calicheamicin, cultured at 37℃and 180rpm until OD600 was 1.0, 1% of the bacterial solution was transferred to 500mL of fresh LB medium containing 100. Mu.g/mL of calicheamicin, cultured at the same conditions until OD600 was 0.6, 25. Mu.L of IPTG at a concentration of 1mol/L was then added to the culture to give a final concentration of 0.5mmol/L of IPTG, and the cells were collected by centrifugation at 4℃and 9000 Xg for 20 minutes, washed 3 times with 100mmol/L of Tris-HCl buffer (pH=8) and finally resuspended to a turbidity (OD 600=30).
The above OD 600=30 cell heavy suspension was subjected to ultrasonic disruption at a low temperature using an ultrasonic cytodisruption apparatus, and then centrifuged at a temperature of 4 ℃ and a centrifugal force of 14000×g for 10 minutes, and the supernatant was collected, which was a crude enzyme solution of Cr (VI) reductase nfnB protein. The crude enzyme solution obtained was further purified using HisPurTM Ni-NTA purification kit to obtain pure enzyme of Cr (VI) reductase nfnB protein.
The analysis of the Cr (VI) reductase nfnB protein before and after purification by SDS-PAGE respectively shows that the detection result is shown in figure 2, wherein a lane M is a protein marker, a lane T1 is Cr (VI) reductase nfnB crude protease, and a lane T2 is Cr (VI) reductase nfnB pure protease.
The concentration of the pure protease of Cr (VI) reductase nfnB was determined using the Bradford kit, and bovine serum albumin was used as a standard, and the target protein (i.e., cr (VI) reductase nfnB protein) was finally detected at a concentration of 1mg/mL.
Example 2
The Cr (VI) reducing activity of the Cr (VI) reductase nfnB protein prepared in the example was verified in this example by the following method:
A standard containing 20mg/L of Cr (VI) was prepared using analytically pure potassium chromate (K 2CrO4), and a standard containing 20mmol/L of NADH was prepared using analytically pure reduced coenzyme I disodium (NADH Na 2).
The total volume of the Cr (VI) reducing activity verification system for the target protein was 1mL, wherein 20mg/L of the Cr (VI) -containing standard was 500. Mu.L, 20mmol/L of the NADH-containing standard was 50. Mu.L, and 450. Mu.L of the Cr (VI) reductase nfnB pure protease solution prepared in example 1 was used, and the final concentration of Cr (VI) in the system was 10mg/L. In addition, an equal volume of sterile water was added as control group 1 (CK 1), and an equal volume of the high-temperature inactivated Cr (VI) reductase nfnB pure enzyme was added as control group 2 (CK 2).
The reduction reaction was carried out at a temperature of 30℃ C, pH and a temperature of 7 for only 5 minutes, and the Cr (VI) remaining in the system was detected by the dibenzoyl dihydrazide method (GB 7467-87). As a result, as shown in FIG. 3, the concentration of Cr (VI) in the treated group to which the pure Cr (VI) reductase nfnB was added was reduced to 5.38mg/L, whereas the concentration of Cr (VI) was not significantly changed in the system to which the pure Cr (VI) reductase nfnB was added with an equal volume of sterilized water and inactivated at high temperature.
Example 3
This example demonstrates the effect of temperature on the reduction activity of Cr (VI) reductase nfnB prepared in example 1 by referring to the system for verifying the reduction activity of Cr (VI) and the method for detecting Cr (VI) in example 2, which was treated at 20, 30, 40, and 50℃for 6 hours, respectively, and then the Cr (VI) remaining in the system was detected.
The relative reduction activity was calculated with respect to the maximum reduction activity, with the temperature having the maximum reduction activity as 100%, and the rest of the reference maximum reduction activities, respectively. As a result, as shown in FIG. 4, cr (VI) reductase nfnB had the maximum activity at 40 ℃, and the activities at 20, 30 and 50℃were 56.79%, 89.69% and 38.14% of the maximum activities, respectively. From this, it was found that Cr (VI) reductase nfnB was active over a wide temperature range.
Example 4
This example demonstrates the effect of pH on the reducing activity of the Cr (VI) reductase nfnB prepared in example 1 by referring to the system for verifying the reducing activity of Cr (VI) and the method for detecting Cr (VI) in example 2, which was treated for 6 hours at pH 6, 7, 8, and 9, respectively, and then detected for Cr (VI) remaining in the system.
The relative reduction activity was calculated with the temperature having the maximum reduction activity as 100% and the remaining reference maximum reduction activity. As a result, as shown in FIG. 5, cr (VI) reductase nfnB had the maximum activity at pH 8, and the activity at pH 7 was 90.01% of the maximum activity, whereas the Cr (VI) reducing activity of Cr (VI) reductase nfnB was almost lost at pH 6 and 9. As can be seen, the Cr (VI) reductase nfnB is relatively specific to the pH requirement.
Example 5
The Cr (VI) reductase nfnB protein prepared in example 1 is used for treating Cr (VI) containing wastewater, and the specific process is as follows:
A certain amount of Cr (VI) containing wastewater is taken from a wastewater field in the part of Wuhan, and the Cr (VI) in the wastewater is detected by using a dibenzoyl dihydrazide method, so that the wastewater contains Cr (VI) and has the concentration of 9.93mg/L.
Cr (VI) reductase nfnB pure enzyme and reduced coenzyme I disodium (NADH Na 2) were added to the above-mentioned Cr (VI) wastewater in the amounts of 50. Mu.g/mL and 1mmol/L, respectively, the reduction reaction was treated at a temperature of 30℃ C, pH for 3 hours, and the residual Cr (VI) concentration in the reaction system was monitored.
As shown in FIG. 6, after the Cr (VI) containing wastewater is treated for 3 hours by using the Cr (VI) reductase nfnB pure enzyme, the concentration of Cr (VI) in the wastewater is reduced from 9.93mg/L to 3.75mg/L, the reduction rate is 62.5%, and the maximum reduction rate of Cr (VI) is 69.2 mu mol.min -1·mg-1, which shows that the enzyme can rapidly and effectively remove hexavalent chromium in the wastewater.
In conclusion, the Cr (VI) reductase provided by the invention can be efficiently expressed by an escherichia coli expression system, can be efficiently and rapidly reduced and removed Cr (VI) in a midbody, and has a wide applicable temperature range, so that the Cr (VI) reductase has a large application potential in Cr (VI) containing wastewater treatment.
The foregoing description of the preferred embodiments of the present invention should not be taken as limiting the scope of the invention, and it should be noted that any modifications, equivalents, improvements and others within the spirit and principles of the present invention will become apparent to those of ordinary skill in the art.
Claims (8)
1. A Cr (VI) reductase is characterized in that the amino acid sequence of the Cr (VI) reductase is shown as SEQ ID NO. 1.
2. The method for producing Cr (VI) reductase according to claim 1, comprising the steps of:
s1, connecting a fragment with a nucleotide sequence shown as SEQ ID NO.3 into a plasmid to obtain a recombinant plasmid, and then converting the recombinant plasmid into escherichia coli BL21 (DE 3) to obtain recombinant bacteria, wherein the recombinant bacteria are induced to express by IPTG;
s2, after the expression is finished, centrifugally collecting cells, carrying out ultrasonic disruption after the cells are resuspended, centrifugally collecting supernatant, and purifying to obtain the pure enzyme of the Cr (VI) reductase.
3. The method of claim 2, wherein the 3' end of the fragment of step S1 is linked to a histidine purification tag and the purification of step S2 is performed using His PurTM Ni-NTA purification kit.
4. A protease preparation, characterized in that the active ingredient of the protease preparation comprises the Cr (VI) reductase according to claim 1.
5. Use of a Cr (VI) reductase as defined in claim 1 or a protease preparation as defined in claim 4 in the remediation of a Cr (VI) contaminated water body.
6. The use according to claim 5, wherein the Cr (VI) reductase is added to a body of water contaminated with Cr (VI) to reduce highly biotoxic, highly water soluble Cr (VI) to low biotoxic, low water soluble Cr (III) to achieve a remediation effect, wherein the reduction conditions are pH 7-8 and temperature 20-50 ℃.
7. The use according to claim 5, wherein the Cr (VI) contaminated water is further dosed with a reduced coenzyme.
8. The use according to claim 7, characterized in that the reduced coenzyme is in particular reduced disodium coenzyme I.
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