CN108060171B - Surface display metal binding protein gene and application thereof in recovery of platinum and palladium metals - Google Patents
Surface display metal binding protein gene and application thereof in recovery of platinum and palladium metals Download PDFInfo
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Abstract
The invention discloses a surface display metal binding protein gene and application thereof in recovering platinum and palladium metals, wherein the nucleotide sequence of the surface display metal binding protein gene is shown as SEQ ID NO. 3; the surface display metal binding protein has an amino acid sequence shown as SEQ ID NO. 6; the recombinant gene engineering bacteria containing the gene are successfully applied to the recovery of the platinum-palladium metal in the industrial wastewater. The EC20 gene and the InaKN gene are connected together through an expression vector pET28a (+) by utilizing the characteristics of the anchoring protein, and are introduced into a microbial receptor for recombinant expression; in the process of recombinant expression, InaKN expresses and fixes EC20 on the surface of the microorganism, thereby improving the selectivity and adsorptivity of the microorganism to platinum-palladium metal; the adsorption capacity of the microorganism to the platinum and palladium metal ions is respectively increased by 1.6 times and 1.3 times, and the recovery rates of the platinum and palladium metal ions in the industrial wastewater respectively reach 100 percent and 97.5 percent.
Description
Technical Field
The invention belongs to the fields of genetic engineering and precious metal recycling, and particularly relates to a surface display metal binding protein gene and application thereof in recycling platinum and palladium metals.
Background
Platinum group metals are widely used in various fields such as industry, agriculture, medicine, catalysis, etc. due to their special physicochemical properties. With the technological progress, the platinum group metals are more widely used and more demanded, but the platinum group metals are low in the earth crust and less than 5% of the platinum group metals are stored in our country, and under the condition, the platinum group metals are recovered from secondary resources, so that the platinum group metals have certain social significance and economic value. At present, methods for recovering platinum group metals from industrial wastewater mainly comprise a chemical precipitation method, an ion exchange method, a solvent extraction method and the like, but the methods are difficult to recover platinum and palladium resources in low-concentration platinum and palladium industrial wastewater.
In recent years, many researchers at home and abroad utilize the characteristics that microorganisms have large specific surface area and various groups on the cell surface are abundant and can adsorb and recover metal ions in a solution, and the microorganisms are applied to adsorbing metal ions such as Cd, Hg, Cu, Ni, Pb, Zn and the like in industrial wastewater, for example, in patent 201210295180.1, Cd in a waste solution is recovered by using endophyte LK92 +、Cu2+、Pb2+Plasma metal ions; however, these microorganisms have low adsorption capacity and poor selectivity for noble metal ions, and thus studies on the recovery of platinum group metal ions by microbial techniques have been reported.
In order to improve the adsorptivity of microorganisms to these metal ions, genetic engineering technology is usually used to introduce protein genes with specific binding function to metal ions into microorganisms for expression, thereby improving the selectivity and adsorptivity of microorganisms to metal ions; for example, in patent 201510762680.5, a new metallothionein gene is introduced into an Escherichia coli receptor to obtain a recombinant genetically engineered bacterium, thereby improving the adsorption effect of the bacterium on heavy metal ions; however, the recombinant gene engineering bacteria obtained by the method have the advantages that the expression of the protein is mainly concentrated in the microbial body, the protein is easily decomposed by protease in the microbial cell, and the stability is poor; and the method is easy to interfere the cell self redox pathway and reduce the activity of the microorganism, so the method has poor stability of the expressed protein and limited improvement on the selectivity and the adsorptivity of the recombinant genetic engineering bacteria.
Disclosure of Invention
The invention aims to provide a surface display metal binding protein gene and application thereof in recovering platinum and palladium metals, and solves the problems of poor selectivity and poor adsorptivity of microorganisms to platinum group metals.
The nucleotide sequence of the surface display metal binding protein gene is shown in SEQ ID NO. 3.
The recombinant plasmid pET28a (+) -InaKN-EC20 contains a gene shown as SEQ ID NO. 3.
The preparation method of the recombinant gene engineering bacterium containing the surface display metal binding protein gene comprises the following steps:
1) artificially synthesizing an anchoring protein (InaKN) gene shown in SEQ ID NO.1 and a metal binding protein (EC20) gene shown in SEQ ID NO. 2;
2) connecting the synthetic sequence in the step 1) to a pET28a (+) expression vector to obtain a recombinant plasmid pET28a (+) -InaKN-EC 20;
3) introducing the recombinant plasmid in the step 2) into a receptor of escherichia coli, performing protein induction expression, and obtaining recombinant genetic engineering bacteria after determining that the expression is correct.
In the step 2), an InaKN gene sequence is inserted between Nco I and Hind III enzyme cutting sites of pET28a (+); the EC20 gene sequence was inserted between Not I and Xho I cleavage sites of pET28a (+).
The recombinant gene engineering bacteria are applied to recovery of platinum and palladium metals.
The use method of the recombinant genetic engineering bacteria for recovering platinum and palladium metals from industrial wastewater comprises the following steps:
1) inoculating the recombinant genetic engineering bacteria into an HB-PET self-induction culture medium, performing activated culture, and centrifugally collecting the thalli of the engineering bacteria after the culture is finished;
2) adding the engineering bacteria thallus in the step 1) into industrial wastewater for adsorption, and after adsorption is finished, centrifugally separating and collecting the thallus.
The principle of the invention is as follows:
the invention connects the metal binding protein (EC20) gene and the anchoring protein (InaKN) gene together through an expression vector pET28a (+), so as to obtain a surface-displayed metal binding protein gene, and obtain a recombinant plasmid pET28a (+) -InaKN-EC20, and then the plasmid is introduced into an escherichia coli receptor to obtain recombinant genetic engineering bacteria; when recombinant genetic engineering bacteria express recombinant plasmids, the N end of the anchoring protein InaKN is connected with the metal binding protein EC20, the other end of the anchoring protein InaKN is connected with aspartic acid residue in a transmembrane-related structural domain, glycosyl phosphatidylinositol on the aspartic acid residue is combined with mannose of outer membrane protein through an N-glycosidic bond, and thus the binding protein is fixed on the surface of a cell outer membrane.
The invention has the beneficial effects that:
the EC20 gene and the InaKN gene are connected together through an expression vector pET28a (+) by utilizing the characteristics of the anchoring protein, and are introduced into a microbial receptor for recombinant expression; in the process of recombinant expression, InaKN expresses and fixes EC20 on the surface of the microorganism, thereby improving the selectivity and adsorptivity of the microorganism to platinum-palladium metal; the adsorption capacity of the microorganism to the platinum and palladium metal ions is respectively increased by 1.6 times and 1.3 times, and the recovery rates of the platinum and palladium metal ions in the industrial wastewater respectively reach 100 percent and 97.5 percent. The recombinant microorganism has the characteristics of easy preparation, low cost, reusability and no secondary pollution, so that the recombinant microorganism has a positive promoting effect on the recycling of precious metal resources.
Drawings
FIG. 1 shows the electrophoresis chart of the surface display metal binding protein expressed by the recombinant genetically engineered bacterium in example 1; m: marker; 1: colibl21 original strain; 2: coli BL21 engineering bacteria;
FIG. 2 is a graph showing the adsorption effect of Escherichia coli on Pd ion solutions of different concentrations in example 2;
FIG. 3 is a graph showing the adsorption effect of E.coli on Pt ion solutions of different concentrations in example 2.
Detailed Description
Example 1
(1) Synthesis of proteins and Metal binding protein genes
The gene sequences for the dockerin (InaKN) and the metal binding protein (EC20) were sent to the organism for synthesis and optimization into gene sequences suitable for expression in E.coli. The synthetic gene sequence was ligated to pET28a (+) plasmid, in which InaKN sequence was inserted between Nco I and Hind III cleavage sites and EC20 sequence was inserted between Not I and Xho I cleavage sites, to give recombinant plasmid pET28a (+) -InaKN-EC20, which contained the nucleotide sequence shown in SEQ ID NO. 3.
(2) Coli BL21 competent cell transformed with recombinant plasmid
Putting 100 mu L of E.coli BL21 competent cells into a centrifuge tube, then putting the centrifuge tube into an ice bath, then adding 10 mu L of recombinant plasmid pET28a (+) -InaKN-EC20 into the centrifuge tube, gently mixing the mixture, and then putting the mixture into the ice bath to stand for 30 min.
② placing the centrifuge tube in 42 ℃ water bath for standing for 90sec, then quickly placing the centrifuge tube in ice bath, and placing for 3min to cool the cells, and the centrifuge tube is not shaken in the process.
③ adding 900 mu L of sterile LB culture medium into the centrifuge tube, repeatedly blowing and sucking by using a pipette gun until the sterile LB culture medium is uniformly mixed, and then placing the mixture into a shaker at 37 ℃ for shaking culture at 170rpm for 45min to enable the thalli to recover.
Fourthly, the content in the centrifuge tube is mixed evenly, then 100 mu L of transformed competent cells are absorbed and added on an LB solid culture medium with kanamycin resistance, a sterile glass coating rod is used for coating evenly, after the liquid is completely absorbed, the flat plate is placed upside down in a constant temperature incubator at 37 ℃ for culturing for 16h, and the transformed bacterial colony is observed.
(3) Inducible expression of proteins
Picking a single transformed colony from the transformed colonies obtained in the step (2), placing the single transformed colony in 10mL of LB liquid medium, and placing the single transformed colony in a shaking table at 37 ℃ for shaking culture at 170rmp for 6 h.
② the culture solution 100 u L inoculation in 10mL LB liquid medium, and adding 50 u L10 mg/mL kanamycin solution, placed in 37 ℃ shaking table 170rpm vibration culture.
③ taking the cultured bacterial liquid, measuring the absorbance of the bacterial liquid at the wavelength of 600nm by using a spectrophotometer, taking out the culture liquid from the shaking table when the OD is 0.5, and cooling.
And fourthly, adding 20 mu L of 100mM IPTG, uniformly mixing, placing the mixture in a shaking table at the temperature of 16 ℃ and at the rpm of 170 for shaking culture for 16h, and obtaining the recombinant bacterium liquid.
(4) Protein electrophoresis
Collecting thalli: taking 1.5mL of E.coli BL21 original bacterial liquid and the recombinant bacterial liquid obtained in the step (3), centrifuging at 10000rpm for 3min, and removing the supernatant. Washed 2 times with 0.01M PBS buffer, centrifuged 3min at 10000rpm, and resuspended in 3mL PBS buffer.
Cell disruption: the centrifuge tube containing the bacterial liquid is placed in an ice bath, and is crushed for 60 times at the time interval of 7 seconds at 240W for 4 seconds/time. And taking out the bacteria liquid after the bacteria liquid becomes clear, and if the bacteria liquid does not become clear, properly increasing the ultrasonic treatment times.
Preparing a sample: sucking 1mL of clarified bacteria liquid, centrifuging at 12000rpm for 3min, sucking 10 μ L of supernatant into a micro centrifuge tube, adding 10 μ L of Loading buffer, sucking by a pipette gun, mixing uniformly, and boiling for 5 min.
And fourthly, adding 10 mu L of protein marker and 20 mu L of boiled sample into each lane of the protein gel by using a pipette gun, and carrying out electrophoresis for 90min at 100V.
Experimental observation: placing the gel in a protein dyeing agent of ProteinShow-G250, boiling for 3min, pouring off the dyeing agent, adding deionized water, boiling for 5min, pouring off waste liquid, adding deionized water, repeating for three times, and placing the gel in a gel imaging system to observe whether the protein is successfully expressed.
The protein electrophoretogram of the recombinant e.coli BL21 in this example is shown in fig. 1, and it can be seen from fig. 1 that: compared with E.coliBL21 original bacteria, the E.coliBL21 recombinant bacteria obviously have a protein band between 25 and 35kDa, which indicates that the surface display metal binding protein gene of the invention is successfully expressed, and the E.coliBL21 recombinant bacteria are obtained.
When recombinant genetic engineering bacteria express recombinant plasmids, the N end of the anchoring protein InaKN is connected with the metal binding protein EC20, the other end of the anchoring protein InaKN is connected with aspartic acid residue in a transmembrane-related structural domain, and the aspartic acid residue is combined with mannose of the outer membrane protein in a mode of N-glycosidic bond of glycosyl phosphatidylinositol, so that the binding protein is fixed on the surface of the outer membrane of the cell.
Example 2
In this example, the palladium (Pd) metal solution was used at a concentration of 0.5, 2.5, 5, 25, 50, 100, 200mg/L and adsorbed at pH 3.0. The specific operation steps of the experiment are as follows (in combination with FIG. 1):
(1) the preparation method of the microbial adsorbent comprises the following specific steps:
coli BL21 stock strain was inoculated into sterilized LB medium, cultured at 30 ℃ for 24 hours at 170rpm, and then centrifuged at 10000rpm for 10min to collect the strain. E.coli BL21 engineered strain prepared in example 1 was inoculated into HB-PET self-induced medium, cultured at 30 ℃ for 24 hours at 170rpm, and then centrifuged at 10000rpm for 10min to collect the cells. The self-induction culture medium is purchased from Qingdao Gaokoubo Biotech Co., Ltd, and the 1L culture medium contains 27.5g of culture powder and 5g of glycerol.
(2) The preparation of the Pd (II) stock solution comprises the following specific steps:
0.834g of PdCl2Dissolving in 500mL deionized water, adding a certain amount of concentrated HCl to adjust the pH value of the solution to 1.0, then placing in an HH-S7S model constant temperature water bath kettle at 80 ℃ and stirring for 1h under the condition of 170rpm until the solution is completely dissolved, cooling, then using a volumetric flask to fix the volume to 1L, thus obtaining 500mg/L Pd (II) stock solution, and using the volumetric flask to dilute the solution to the required concentration.
(3) The biological adsorption experiment comprises the following specific steps:
pd (ii) metal solution with pH3.0 and concentration of 0.5, 2.5, 5, 25, 50, 100, 200mg/L was prepared, and e.coli BL21 original strain or recombinant genetically engineered strain was added at final concentration of 5 g/L. Mixing the two solutions, adsorbing at 30 deg.C and 170rpm for 3h, centrifuging at 10000rpm for 5min, and measuring the concentration of Pd (II) remaining in the supernatant by atomic absorption method.
The experimental result is shown in fig. 2, the adsorption capacity of the modified e.coli BL21 to palladium ions is obviously high, and the adsorption capacity is increased from 109.334mg/g to 143.768mg/g, which is increased by about 1.3 times. The whole adsorption process is very rapid, and the adsorption capacity reaches more than 90 percent of the total adsorption capacity within 30 min. Therefore, the gene modification can effectively improve the adsorption capacity of E.coli BL21 on palladium ions, has short adsorption time and has huge industrial application potential.
Example 3
In this example, the platinum (Pt) metal solution was used at concentrations of 1, 5, 10, 50, 100, 200mg/L and an adsorption pH of 3.0. The specific operation steps of the experiment are as follows:
(1) the preparation method of the microbial adsorbent comprises the following specific steps:
coli BL21 stock strain was inoculated into sterilized LB medium, cultured at 30 ℃ for 24 hours at 170rpm, and then centrifuged at 10000rpm for 10min to collect the strain. E.coli BL21 engineering bacteria prepared in example 1 were inoculated into HB-PET self-induction medium, cultured at 30 ℃ for 24 hours at 170rpm, and then centrifuged at 10000rpm for 10min to collect the cells. The self-induction culture medium is purchased from Qingdao Gaokoubo Biotech Co., Ltd, and the 1L culture medium contains 27.5g of culture powder and 5g of glycerol.
(2) The preparation method of the Pt (II) stock solution comprises the following specific steps:
0.864g of PtCl2Dissolving in 500mL deionized water, shaking at 170rpm for 1h to dissolve completely, diluting to 1L with volumetric flask to obtain 500mg/L Pt (IV) stock solution, and diluting to desired concentration with volumetric flask when in use.
(3) The biological adsorption experiment comprises the following specific steps:
pt (iv) metal solution with pH3.0 and concentration of 1, 5, 10, 50, 100, 200mg/L was prepared, and e.coli BL21 raw or engineered bacteria was added at a final concentration of 5 g/L. After mixing them well, adsorbing at 30 deg.C and 170rpm for 3h, centrifuging at 10000rpm for 5min, and measuring the concentration of residual Pt (IV) in the supernatant by atomic absorption method.
The experimental result is shown in fig. 3, the adsorption capacity of the e.coli BL21 to platinum ions is only 69.630mg/g, and after the gene modification, the adsorption capacity of the e.coli BL21 engineering bacteria to platinum ions reaches 112.669mg/g, which is 1.6 times of the former. In addition, the adsorption process takes a short time, and the adsorption process is basically balanced in 90 min. Therefore, the engineering bacteria can be used for adsorbing and recovering platinum ions in the solution and have industrial application potential.
Example 4
In the embodiment, an industrial wastewater solution is adopted, and the specific operation steps of the experiment are as follows:
(1) the preparation method of the microbial adsorbent comprises the following specific steps:
coli BL21 stock strain was inoculated into sterilized LB medium, cultured at 30 ℃ for 24 hours at 170rpm, and then centrifuged at 10000rpm for 10min to collect the strain. E.coli BL21 engineered strain prepared in example 1 was inoculated into HB-PET self-induced medium, cultured at 30 ℃ for 24 hours at 170rpm, and then centrifuged at 10000rpm for 10min to collect the cells. The self-induction culture medium is purchased from Qingdao Gaokoubo Biotech Co., Ltd, and the 1L culture medium contains 27.5g of culture powder and 5g of glycerol.
(2) The biological adsorption experiment comprises the following specific steps:
adding 5g/L E.coli BL21 raw bacteria or engineering bacteria into the industrial wastewater raw solution, mixing the two, adsorbing for 3h at 30 ℃ and 170rpm, centrifuging for 5min at 10000rpm, and analyzing all elements in the supernatant by an atomic absorption method. Meanwhile, analyzing the types and contents of all elements in the industrial wastewater before adsorption.
The experimental results are shown in table 1, wherein the industrial wastewater stock solution contains 19 elements in total, the highest content of the elements is Al, Na and Fe, and the concentrations of the elements reach 10350, 1095.5 and 133.2mg/L respectively. In addition, the content of Sn and Ca ions in the industrial wastewater is also high, and the concentration is 28.76 and 23.95 mg/L. The concentrations of the noble metals Pd, Pt and Ir reach 24.51, 3.21 and 5.84mg/L, and the method has high recovery value. The result shows that the E.coli BL21 raw bacteria and the engineering bacteria have strong selective adsorption on Pd and Pt in the wastewater, after adsorption for 3 hours, the Pd concentrations in the supernatant of the adsorption wastewater of the raw bacteria and the engineering bacteria are only 2.99 and 0.627mg/L, and the adsorption rates reach 88.2 percent and 97.5 percent. For Pt, the concentration of residual metal in the solution after the original E.coli BL21 bacteria are adsorbed is 0.218mg/L, and the modified E.coli BL21 can completely adsorb Pt metal in industrial wastewater, and the adsorption rate reaches 100%. Although the concentrations of Al and Fe ions in the industrial wastewater are high, experimental results show that the E.coli BL21 raw bacteria and the engineering bacteria have little adsorption on the raw bacteria and the engineering bacteria and also have little adsorption on other metal ions, which shows that the strains have selective adsorption on Pd and Pt ions in the industrial wastewater and can be used for recovering Pd and Pt noble metals in the industrial wastewater.
Table 1 e. adsorption of raw and engineered bacteria BL21 on industrial wastewater
Sequence listing
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Claims (3)
1. The application of recombinant gene engineering bacteria containing surface display metal binding protein genes in recovery of platinum and palladium metals is characterized in that:
the nucleotide sequence of the surface display metal binding protein gene is shown as SEQ ID NO. 3;
the preparation method of the recombinant gene engineering bacteria containing the surface display metal binding protein gene comprises the following steps:
1) artificially synthesizing an anchoring protein gene shown in SEQ ID NO.1 and a metal binding protein gene shown in SEQ ID NO. 2;
2) connecting the synthesized sequence metal binding protein gene and the anchored protein gene in the step 1) to a pET28a (+) expression vector to construct a recombinant expression plasmid pET28a (+) -InaKN-EC 20;
3) introducing the recombinant plasmid obtained in the step 2) into a receptor of escherichia coli, performing protein induction expression, and obtaining recombinant genetic engineering bacteria containing a surface display metal binding protein gene after determining that the expression is correct;
the recombinant expression plasmid pET28a (+) -InaKN-EC20 in the step 2) comprises a nucleotide sequence of a surface display metal binding protein gene shown in SEQ ID NO. 3.
2. The application of the recombinant gene engineering bacteria containing the surface display metal binding protein gene in the recovery of platinum-palladium metal is characterized in that in the step 2), an anchoring protein gene sequence is inserted between Nco I and Hind III enzyme cutting sites of pET28a (+); the metal binding protein gene sequence was inserted between Not I and Xho I cleavage sites of pET28a (+).
3. The application of recombinant genetic engineering bacteria containing surface display metal binding protein genes in recovery of platinum and palladium metals is characterized in that the method for adsorbing and recovering the platinum and palladium metals in industrial wastewater by the recombinant genetic engineering bacteria comprises the following steps:
1) inoculating the recombinant genetic engineering bacteria into an HB-PET self-induction culture medium, performing activated culture, and centrifugally collecting the thalli of the engineering bacteria after the culture is finished;
2) adding the engineering bacteria thallus in the step 1) into industrial wastewater for adsorption, and after adsorption is finished, centrifugally separating and collecting the thallus.
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| CN201711439270.2A CN108060171B (en) | 2017-12-27 | 2017-12-27 | Surface display metal binding protein gene and application thereof in recovery of platinum and palladium metals |
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| CN201711439270.2A CN108060171B (en) | 2017-12-27 | 2017-12-27 | Surface display metal binding protein gene and application thereof in recovery of platinum and palladium metals |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006072845A2 (en) * | 2004-12-02 | 2006-07-13 | Csir | Gram positive bacterial cells comprising a disrupted flagellin gene, flagellin-based fusion proteins and use in removal of metal ions from a liquid |
| CN103275919A (en) * | 2013-06-18 | 2013-09-04 | 江苏省农业科学院 | Escherichia coli engineering bacteria and preparation method and applications thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006072845A2 (en) * | 2004-12-02 | 2006-07-13 | Csir | Gram positive bacterial cells comprising a disrupted flagellin gene, flagellin-based fusion proteins and use in removal of metal ions from a liquid |
| CN103275919A (en) * | 2013-06-18 | 2013-09-04 | 江苏省农业科学院 | Escherichia coli engineering bacteria and preparation method and applications thereof |
Non-Patent Citations (2)
| Title |
|---|
| Cell surface display of synthetic phytochelatins using ice nucleation protein for enhanced heavy metal bioaccumulation;WeonBae等;《Journal of Inorganic Biochemistry》;20020115;第88卷(第2期);第223页摘要，第224页左栏"2.1 Strains, plasmids, media, and general procedures"部分、"2.2 Construction of ice-nucleation fusions"部分，第225页左栏第1段 * |
| Functional Display of Foreign Protein on Surface of Escherichia coli Using N-Terminal Domain of Ice Nucleation Protein;Lin Li等;《Biotechnol Bioeng》;20040120;第85卷(第2期);第214页摘要 * |
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