Preparation method and application of a polypeptide for absorbing and enriching hexavalent chromium ions and a carrier thereof

Abstract

The application discloses a kind of hexavalent chromium ion adsorption enrichment polypeptide and its carrier preparation method and application.The carrier is prepared by the following method: 200mg of gamma-Fe2O3 Nanoparticle is dispersed in 4mL of water,800mg chitosan is stirred uniformly,then 4mL of glutaraldehyde is added.The dispersion is stirred at 45 DEG C for 3 hours.Supernatant is poured out, and the solid is washed with distilled water and dried.The immobilization of polypeptide and carrier is prepared by the following method: 10mg polypeptide is dissolved in phosphate solution.100mg of carrier is weighed and added, the mixture is magnetically stirred at room temperature for 7 hours, the obtained solid is washed with phosphate solution for 3 times, then air dried at 37-38 DEG C to obtain hexavalent chromium ion adsorbent.The hexavalent chromium ion adsorbent of the application can selectively and highly adsorb hexavalent chromium, and then eluted by dilute nitric acid to effectively enrich hexavalent chromium from liquid.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and in particular to a method for preparing and applying a hexavalent chromium ion-adsorbed enriched polypeptide and its carrier. Background Technology

[0002] Chromium is a naturally occurring metallic element found in rocks, soil, volcanic ash, and gases, as well as in plants and animals. The most common forms of this element are the metallic forms chromium(O), trivalent chromium (Cr(III)), and hexavalent chromium (Cr(VI)). Hexavalent chromium is a known toxin, mutagen, and carcinogen. Trivalent chromium was identified as an essential trace element for mammals more than 50 years ago, necessary for normal glucose metabolism. In fact, chromium deficiency in humans can lead to symptoms associated with type II diabetes and cardiovascular disease. Chromium is a metal oxyanion chromate (CrO4). 2- Cr(VI) is a widespread environmental pollutant due to its extensive use in industrial and defense sectors, such as leather tanning, electroplating, paint and pigment manufacturing, stainless steel welding, and nuclear weapons production. Cr(VI) is highly soluble and toxic; it can enter the human body through the digestive tract, respiratory tract, skin, and mucous membranes. Long-term exposure can lead to mutations and cancer. For example, Cr(VI) exposure in cultured cells causes a range of genomic damage, including DNA single- and double-strand breaks, amino acid and protein binding to DNA, DNA interstrand cross-linking, and Cr-DNA adducts. Cr toxicity is also associated with reactive oxygen species generated during the partial reduction of Cr(VI) to unstable intermediate Cr(V) by various in vivo non-specific reducing agents (such as glutathione, NADH, NADPH, and cysteine) or cellular single-electron reductases.

[0003] Traditional methods for detecting hexavalent chromium require the extraction of hexavalent chromium through strong acid, high-temperature digestion, or extraction before instrumental detection. Based on this, this invention proposes a method for the adsorption, enrichment, and preparation of hexavalent chromium ions. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for adsorbing and enriching polypeptides by hexavalent chromium ions.

[0005] Another objective of this invention is to provide a hexavalent chromium ion adsorption and enrichment polypeptide carrier.

[0006] Another objective of this invention is to provide a method for preparing a polypeptide carrier by adsorption and enrichment of hexavalent chromium ions.

[0007] Another object of the present invention is to provide applications of the above-mentioned polypeptides and polypeptide carriers.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A polypeptide for adsorbing and enriching hexavalent chromium ions, comprising at least one of the following polypeptides:

[0010] Cbp-1, whose amino acid sequence is NHIYGRTTPEWR;

[0011] Cbp-2, whose amino acid sequence is YSDSTSAMRGLM;

[0012] Cbp-3, whose amino acid sequence is LGVANPLRSATD;

[0013] Cbp-4, whose amino acid sequence is IYRSDTSCKMCE;

[0014] Cbp-5, whose amino acid sequence is GFFQVPGQHVNK;

[0015] Cbp-6, whose amino acid sequence is DYKTHGFPALRH;

[0016] Cbp-7, whose amino acid sequence is HFVKTPARWAWG;

[0017] Cbp-8, whose amino acid sequence is FIGLP VITEIPF;

[0018] Cbp-9 has the amino acid sequence YGNAHIARLLVP.

[0019] Preferably, the hexavalent chromium ion adsorption enrichment polypeptide is at least one of Cbp-1, Cbp-4, Cbp-6 and Cbp-7.

[0020] Preferably, the hexavalent chromium ion adsorption enrichment polypeptide is at least one of Cbp-6 and Cbp-7.

[0021] A method for preparing a polypeptide carrier for adsorption and enrichment by hexavalent chromium ions includes the following steps:

[0022] (1) Disperse γ-Fe2O3 nanoparticles in water, add chitosan and mix well, then add glutaraldehyde, stir and dry to obtain nano carrier;

[0023] (2) Dissolve the hexavalent chromium ion-adsorbed and enriched polypeptide in the solution, add the nano carrier prepared in step (1), stir, filter, wash and dry to obtain the hexavalent chromium ion-adsorbed and enriched polypeptide carrier.

[0024] The mass ratio of γ-Fe2O3 nanoparticles to chitosan in step (1) is 1:2 to 8; preferably 1:4.

[0025] The amount of water mentioned in step (1) is based on the amount that can dissolve the solute.

[0026] The ratio of glutaraldehyde to γ-Fe2O3 nanoparticles in step (1) is 1 mL: 30-100 mg; preferably 1 mL: 50 mg.

[0027] The stirring conditions described in step (1) are stirring at 40-50°C for 2-4 hours; preferably at 45°C for 3 hours.

[0028] The mass ratio of the hexavalent chromium ion adsorption enrichment peptide to the nano carrier in step (2) is 1:5 to 20; preferably 1:10.

[0029] The solution described in step (2) is a phosphate solution.

[0030] The stirring conditions described in step (2) are 800-900 rpm for 6-8 hours.

[0031] The hexavalent chromium ion adsorption and enrichment polypeptide carrier prepared by the above preparation method.

[0032] The above-mentioned hexavalent chromium ion adsorption enrichment peptides and their carriers are used in the preparation of hexavalent chromium adsorbents.

[0033] The above-mentioned hexavalent chromium ion adsorption enrichment peptides and their carriers are used in the adsorption of hexavalent chromium.

[0034] The present invention has the following advantages and effects compared with the prior art:

[0035] 1. The preparation of the hexavalent chromium ion adsorption enrichment polypeptide described in this invention is simple and can rapidly adsorb hexavalent chromium ions in solution.

[0036] 2. By loading peptides onto a magnetic core, it is possible to quickly recover and separate hexavalent chromium ions from water.

[0037] 3. Because the carrier is loaded, it can be reused multiple times. It can selectively and highly adsorb hexavalent chromium, and then effectively enrich hexavalent chromium from the liquid by elution with dilute nitric acid. Attached Figure Description

[0038] Figure 1 The affinity of nine polypeptides and the original peptide library for hexavalent chromium ions.

[0039] Figure 2 The affinity of four polypeptides with high affinity for hexavalent chromium for different metal ions.

[0040] Figure 3 The adsorption capacity per unit of each adsorbent in Example 2.

[0041] Figure 4 The amount of each adsorbent removed in Example 2.

[0042] Figure 5 The release rate of each adsorbent in Example 2. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0044] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0045] Example 1: Screening for polypeptide sequences that specifically bind to hexavalent chromium

[0046] (1) Preparation of metal complex resin. A hexavalent chromium ion immobilization resin was prepared by replacing pre-loaded nickel ions with hexavalent chromium ions on Ni-NTA Sefinose (Bio Basic Inc., Canada). Specifically, to strip Ni(II) from the Ni-NTA-Sefinose resin, the resin beads were first washed with sufficient EDTA solution (0.5 mol / L, pH 8.0) until the resin changed from blue to colorless. Then, it was rinsed three times with citrate buffer (pH 4.4) and stored at 4°C for future use. This metal-free resin was labeled M. - Resin. To prepare hexavalent chromium-fixed resin, 1 mL of M... - The resin suspension was incubated overnight in 5 mL of hexavalent chromium solution (K₂Cr₂O₇, 1.0 mmol / L) with gentle shaking. It was then washed six times with citrate buffer (pH 4.4) to obtain a metal complex resin containing K₂Cr₂O₇, which was stored at 4°C. Resins loaded with other metals were prepared using a similar process, except that the metal salt (M...) was used instead of... + The metal complex resin containing PbSO4, CdCl2, HgCl2, Cr(NO3)3, and Na3AsO4 is obtained by replacing PbSO4, CdCl2, HgCl2, Cr(NO3)3, and Na3AsO4 metal ions.

[0047] (2) Add 10 μL of phage culture (phage random dodecapeptide library, New England Biolabs, USA, titer approximately 10). 13The phage (pfu / mL) was dissolved in 500 μL of TBS buffer (8 g NaCl, 3 g Tris base, 800 mL water, pH adjusted to 7.4 with concentrated hydrochloric acid, then diluted to 1000 mL with water). 100 μL (sedimentation volume) of resin loaded with hexavalent chromium was added. The mixture was incubated at 25°C and 100 rpm for 25 min, then allowed to stand and the supernatant was discarded. The resin was then washed 8 times with 1 mL of 0.1% TBST buffer (TBS with 0.1% Tween-20 added) to remove loosely bound portions. Finally, 1 mL of 0.5 M EDTA elution buffer was added, and the mixture was incubated at 25°C and 200 rpm for 10 min. The eluent was collected. The retained phage was dissolved in 600 μL of TBS, and the titer was determined and amplified to the desired titer (greater than 10). 13 (pfu / mL, using conventional phage amplification methods), and then proceed to the next round of screening according to the methods described above.

[0048] Three rounds of screening were repeated to obtain nine phage strains with high affinity for hexavalent chromium. These strains were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the polypeptide sequences displayed on their surfaces are shown in Table 1.

[0049] Table 1. Peptide sequences on the surface of nine bacteriophages with high affinity for hexavalent chromium.

[0050]

[0051] (3) The binding affinity and selectivity were further evaluated using ELISA, and the titers of the nine amplified phages and the original peptide library were uniformly adjusted to the order of magnitude of 10. 11 Pfu / mL, add 1 mL of PBS (pH 5.8) buffer to 100 μL of phage suspension, then add 200 μL (sedimentation volume) of metal complexing resin solution containing K2Cr2O7 (metal ion concentration of 0.1 mol / L). Shake for 1 h at 25℃ and 150 rpm. Wash three times with 1 mL of PBS (pH 5.8) buffer. Add 1 mL of HRP-labeled M13 antibody (1:5000 diluted in pH 5.8 PBS buffer). Shake for 1 h at 25℃ and 150 rpm. Wash three times with 1 mL of PBS buffer containing 0.5% Tween 20. Add 400 μL of HRP substrate solution (22 mg ABTS dissolved in 100 mL of 0.2 mol / L pH 5.8 PBS solution). Shake for 1 h at 25℃ and 150 rpm. Measure the absorbance at 405 nm using a microplate reader. Experimental results are as follows: Figure 1 As shown, the affinity of nine phage surface-displayed peptides and the original peptide library for hexavalent chromium was obtained.

[0052] Then, the four highest-valued peptides displayed on the surface of the bacteriophages were selected, and their affinity for PbSO4, CdCl2, HgCl2, Cr(NO3)3, and Na3AsO4 metal ions (metal ion concentration of 0.1 mol / L) was tested using the method described above. The results are as follows: Figure 2 As shown, it can be seen that peptides with a high affinity for hexavalent chromium also have a high affinity for trivalent chromium.

[0053] Example 2: Peptide Immobilization and Adsorption / Desorption Enrichment

[0054] (1) Peptides Cbp-6 and Cbp-7 with good affinity for hexavalent chromium were obtained through screening in Example 1. Cbp-6 and Cbp-7 sequence peptides were then synthesized by Shanghai Sangon Biotech Co., Ltd. First, a carrier for immobilizing the peptides was prepared. 200 mg of γ-Fe2O3 nanoparticles (Maclean, 20 nm spherical, 99.5%, γ-type) were dispersed in 4 mL of water, and 800 mg of chitosan was added and vortexed. Then, 4 mL of glutaraldehyde was added. The dispersion was stirred at 45 °C for 3 hours. The supernatant was poured off, and the solid was washed with distilled water and dried to obtain the carrier. 10 mg of the peptide was weighed and dissolved in a phosphate (pH 7.0) solution. 100 mg of the carrier was then weighed and added to the solution. The mixture was magnetically stirred at room temperature (800–900 rpm) for 7 hours. The obtained solid was washed three times with a phosphate (pH 7.0) solution and then air-dried at 37–38 °C to obtain the fully loaded carrier. Repeat the above steps to obtain Cbp-6-nano adsorbent loaded with Cbp-6, Cbp-7-nano adsorbent loaded with Cbp-7, and adsorbent without peptide modification of nano.

[0055] (2) Prepare a 100 μg / mL Cr(VI) standard stock solution. Weigh 0.2829 g of potassium dichromate, dry it at 120℃ for 2 h, dissolve it in water, transfer it to a 1000 mL volumetric flask, and dilute it to the mark with water. Shake well. Pipette 0.1 mL (10 μg) and 0.05 mL (5 μg) of Cr(VI) standard stock solution into 2 mL centrifuge tubes respectively. Weigh 10 mg of adsorbent into each centrifuge tube, add phosphate (pH 7.0) to 1.5 mL, shake at 25℃ and 200 rpm for 4 hours, centrifuge, and take the supernatant. Detect the hexavalent chromium concentration according to the literature (Yao Jianting, Chen Junjun, Chen Rong. Study on rapid determination of chromium ion concentration [J]. Monitoring and Analysis, 2007, 1005-4944). Figure 3 The adsorption capacity of each adsorbent was obtained. The experimental results show that the support itself also has a small amount of adsorption capacity, but Cbp-6-nano and Cbp-7-nano have a higher adsorption capacity of about 10 μmol Cr(VI) / g adsorbent in high concentration hexavalent chromium solution, which is much higher than that of unsupported nano adsorbent.

[0056] (3) Wash the adsorbent solid from step (2) three times with phosphate (pH 7.0) solution, add 0.1M dilute nitric acid to a final volume of 1.5 mL, and desorb by shaking at 200 rpm for 20 minutes at 25°C. Centrifuge and collect the supernatant to determine the hexavalent chromium concentration. Figure 4 The amount of material removed was calculated. Figure 5 The extraction rate, as demonstrated by the experimental results, is approximately 65% ​​of the adsorbed Cr(VI) can be extracted.

[0057] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for adsorbing and enriching polypeptides by hexavalent chromium ions, characterized in that... The following polypeptides are included: Cbp-6, whose amino acid sequence is DYKTHGFPALRH.

2. A method for preparing a polypeptide carrier for adsorption and enrichment by hexavalent chromium ions, characterized in that... Includes the following steps: (1) Disperse γ-Fe2O3 nanoparticles in water, add chitosan and mix well, then add glutaraldehyde, stir and dry to obtain nano carrier; (2) Dissolve the hexavalent chromium ion adsorption enriched polypeptide in the solution, add the nano carrier prepared in step (1), stir, filter, wash and dry to obtain the hexavalent chromium ion adsorption enriched polypeptide carrier. The hexavalent chromium ion adsorption and enrichment polypeptide is Cbp-6, and its amino acid sequence is DYKTHGFPALRH; The mass ratio of γ-Fe2O3 nanoparticles to chitosan in step (1) is 1:2 to 8; The ratio of glutaraldehyde to γ-Fe2O3 nanoparticles in step (1) is 1 mL: 30-100 mg; The amount of water mentioned in step (1) is based on the amount that can dissolve the solute; The stirring conditions described in step (1) are stirring at 40-50°C for 2-4 hours; The mass ratio of the hexavalent chromium ion adsorption enrichment peptide to the nano carrier in step (2) is 1:5 to 20; The solution described in step (2) is a phosphate solution; The stirring conditions described in step (2) are 800-900 rpm for 6-8 hours.

3. A carrier for adsorbing and enriching polypeptides by hexavalent chromium ions, characterized in that... It is prepared by the preparation method described in claim 2.

4. The application of the hexavalent chromium ion adsorption and enrichment polypeptide according to claim 1 or the carrier according to claim 3 in the preparation of hexavalent chromium adsorbent.

5. The application of the hexavalent chromium ion adsorption and enrichment polypeptide according to claim 1 or the carrier according to claim 3 in the adsorption of hexavalent chromium.

Images