Preparation and application of composite heavy metal adsorbent

Abstract

The invention provides preparation and application of a composite heavy metal adsorbent, and belongs to the technical field of protein engineering. The invention provides a fusion protein. The fusion protein comprises CBM and metallothionein which are sequentially connected from a C terminal to an N terminal, and the metallothionein comprises MTT1, MTT5, TM1 or TM2. According to the invention, MTT1, MTT5, TM1 and TM2 have heavy metal ion binding preference; the CBM has excellent cellulose adsorption performance. The fusion protein is constructed by utilizing CBM and metallothionein, and the fusion protein can be combined with cellulose to modify the cellulose, so that the compound heavy metal adsorbent with an excellent effect is prepared, and pollution caused by heavy metal ions Cd < 2 + > and Pb < 2 + > is effectively treated.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering, and in particular relates to the preparation and application of a composite heavy metal adsorbent. Background Art

[0002] Heavy metal pollution has a huge impact on the entire ecological environment, and heavy metal pollution in the water environment is directly related to human health. The main heavy metals in polluted water include copper, zinc, cadmium, lead, nickel, etc. Among them, cadmium and lead are non-essential elements. Cadmium is the most toxic heavy metal in the aquatic environment, and lead has no known biochemical benefits to the human body. Both should be completely removed from the polluted environment.

[0003] Carbohydrates are biomolecules involved in many life processes. The region in carbohydrate-active enzymes that provides carbohydrate binding affinity is called the carbohydrate binding module (CBM), and the CBM is an excellent model system to study the mechanism of protein-carbohydrate recognition.

[0004] Metallothioneins (MTs) are widely present in various organisms. They are a class of low molecular weight proteins rich in cysteine ​​(Cys) and can be induced to express by metals such as cadmium, copper, zinc, lead or mercury.

[0005] There is currently no report on the construction of composite heavy metal adsorbents based on the combination of CBM and protozoan metallothionein. Summary of the invention

[0006] The purpose of the present invention is to provide the preparation and application of a composite heavy metal adsorbent. The fusion protein of the present invention can be combined with cellulose to construct a composite heavy metal adsorbent.

[0007] The present invention provides a fusion protein, comprising a CBM and a metallothionein connected sequentially from the C-terminus to the N-terminus; the metallothionein comprises MTT1, MTT5, TM1 or TM2; the amino acid sequence of the MTT1 is shown in SEQ ID NO.1; the amino acid sequence of the MTT5 is shown in SEQ ID NO.2; the amino acid sequence of the TM1 is shown in SEQ ID NO.3; the amino acid sequence of the TM2 is shown in SEQ ID NO.4; and the amino acid sequence of the CBM is shown in SEQ ID NO.5.

[0008] The present invention also provides a nucleic acid molecule encoding the fusion protein described in the above scheme.

[0009] The present invention also provides a recombinant vector into which the nucleic acid molecule described in the above scheme is inserted.

[0010] The present invention also provides a host cell comprising the recombinant vector described in the above scheme.

[0011] The present invention also provides a composite heavy metal adsorbent, which uses cellulose as a matrix and adsorbs the fusion protein described in the above scheme.

[0012] The present invention also provides a method for preparing the composite heavy metal adsorbent described in the above scheme, comprising the following steps: mixing cellulose and a solution containing the fusion protein described in the above scheme, and adsorbing the cellulose to obtain the composite heavy metal adsorbent.

[0013] The present invention also provides the use of the fusion protein, the nucleic acid molecule, the recombinant vector, the host cell, the composite heavy metal adsorbent or the composite heavy metal adsorbent prepared by the preparation method described in the above scheme in at least one of the following: 1) heavy metal adsorption; 2) heavy metal enrichment; 3) heavy metal pollution control.

[0014] Preferably, the heavy metals include Pb 2+ and / or Cd 2+ .

[0015] The present invention also provides a method for heavy metal adsorption or heavy metal enrichment, comprising the following steps:

[0016] The composite heavy metal adsorbent described in the above scheme or the composite heavy metal adsorbent prepared by the preparation method is mixed with an article containing heavy metals for adsorption.

[0017] The present invention also provides a method for treating heavy metal pollution, comprising the following steps: applying the composite heavy metal adsorbent described in the above scheme or the composite heavy metal adsorbent prepared by the preparation method to the soil or water body to be treated for adsorption.

[0018] The present invention provides a fusion protein, comprising a CBM and a metallothionein connected sequentially from the C-terminus to the N-terminus; the metallothionein comprises MTT1, MTT5, TM1 or TM2. In the present invention, MTT1, MTT5, TM1 and TM2 have a preference for heavy metal ion induction, wherein the cellulose composite adsorbent derived from MTT1 binds more Cd 2 , while the cellulose composite adsorbents from MTT5, TM1, and TM2 bound more Pb 2+ Ions; CBM has excellent cellulose adsorption performance. The present invention uses CBM and metallothionein to construct a fusion protein, which can bind to cellulose and modify cellulose, thereby preparing an excellent composite heavy metal adsorbent, which can effectively treat heavy metal ions Cd 2+ , Pb 2+ pollute. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0020] Figure 1 The experimental results of the soakability and reusability of cellulose materials are shown in Figure 1. (a) 12 hours is a cycle of repeated soaking and drying of cellulose materials, A: soaking for 12 hours; B: drying for 12 hours; C: soaking for 12 hours; D: drying for 12 hours; E: soaking for 12 hours; F: drying for 12 hours; (b) 72 hours of continuous soaking and drying of cellulose materials, A1: 0 hours; B1: soaking for 12 hours; C1: soaking for 24 hours; D1: soaking for 48 hours; E1: soaking for 72 hours; F1: drying for 24 hours.

[0021] Figure 2 Schematic diagram of cellulose adsorption fusion protein;

[0022] Figure 3The results of SDS-PAGE analysis of SUMO-CBM-MTT1 and GST-CBM-MTT1 binding to cellulose; wherein, (a): SDS-PAGE analysis of SUMO-CBM-MTT1 binding to cellulose, Lane M: protein marker; Lane 1: E. coli BL21 / pET-28a-SUMO 37℃ uninduced sample; Lane 2: E. coli BL21 / pET-28a-SUMO 37℃ 0.1mM IPTG induced sample; Lane 3: E. coli BL21 / pSUMO-CBM-MTT137℃ uninduced whole bacteria sample; Lane 4: E. coli BL21 / pSUMO-CBM-MTT137℃ uninduced supernatant sample (before adsorption); Lane 5: E. coli BL21 / pSUMO-CBM-MTT137℃ uninduced supernatant (after adsorption); Lane 6: supernatant after cellulose binding protein desorption; (b): SDS-PAGE analysis of GST-CBM-MTT1 binding ability to cellulose, Lane M: protein marker; Lane 1: E. coli BL21 / pGEX-6P-137℃ uninduced sample; Lane 2: E. coli BL21 / pGEX-6P-137℃0.1mM IPTG induced sample; Lane 3: E. coli BL21 / pGST-CBM-MTT137℃ uninduced supernatant sample; Lane 4: E. coli BL21 / pGST-CBM-MTT137℃0.2mM IPTG uninduced supernatant sample (before adsorption); Lane 5: E. coli BL21 / pGST-CBM-MTT137℃0.2mM IPTG induced supernatant (after adsorption);

[0023] Figure 4The results of SDS-PAGE analysis of the binding efficiency of GST-CBM-MTT1 to cellulose; wherein, (a): SDS-PAGE analysis of the cellulose adsorption effect of GST-CBM-MTT1 at different volumes, Lane M: protein marker; Lane 1: E. coli BL21 / pGEX-6P-137℃ 0.1mM IPTG induced sample; Lane 2: E. coli BL21 / pGST-CBM-MTT137℃ uninduced sample; Lane 3: E. coli BL21 / pGST-CBM-MTT137℃ 0.2mM IPTG uninduced supernatant sample (before adsorption); Lanes 4 to 7: E. coli BL21 / pGST-CBM-MTT137℃ 0.2mM IPTG induced 1mL, 2mL, 3mL, 4mL supernatant (after adsorption); (b): SDS-PAGE analysis of GST-CBM-MTT1 cellulose desorption at different volumes, Lane M: protein marker; Lane 1: E. coli BL21 / pGEX-6P-137℃0.1mM IPTG-induced sample; Lane 2: E. coli BL21 / pGST-CBM-MTT137℃0.2mM IPTG-uninduced supernatant sample (before adsorption); Lane 3-6: E. coli BL21 / pGST-CBM-MTT137℃0.2mM IPTG-induced 1mL, 2mL, 3mL, 4mL supernatant (desorption);

[0024] Figure 5The results of SDS-PAGE analysis of the binding ability of SUMO-CBM-TM1 and GST-CBM-TM1 to cellulose; wherein, (a): SDS-PAGE analysis of the binding ability of SUMO-CBM-TM1 to cellulose, Lane M: protein marker; Lane 1: E. coli BL21 / pET-28a-SUMO 37℃ 0.1mM IPTG induced sample; Lane 2: E. coli BL21 / pSUMO-CBM-TM137℃ uninduced supernatant sample; Lane 3: E. coli BL21 / pSUMO-CBM-TM116℃ 0.1mM IPTG induced whole bacteria sample; Lane 4: E. coli BL21 / pSUMO-CBM-TM116℃ 0.1mM IPTG induced supernatant sample (before adsorption); Lane 5: E. coli BL21 / pSUMO-CBM-TM116℃ 0.1mM IPTG-induced supernatant sample (after adsorption); Lane 6: supernatant after desorption of cellulose-bound SUMO-CBM-TM1; (b): SDS-PAGE analysis of the binding ability of GST-CBM-TM1 to cellulose, Lane M: protein marker; Lane 1: E. coli BL21 / pGEX-4T-137℃0.1mM IPTG-induced sample; Lane 2: E. coli BL21 / pGST-CBM-TM137℃ uninduced supernatant sample; Lane 3: E. coli BL21 / pGST-CBM-TM137℃0.1mMIPTG-induced whole bacteria sample; Lane 4: E. coli BL21 / pGST-CBM-TM137℃0.1mM IPTG-induced supernatant sample (before adsorption); Lane 5: E. coli BL21 / pGST-CBM-TM137℃0.1mM IPTG-induced supernatant sample (after adsorption); Lane 6: supernatant after desorption of cellulose-bound GST-CBM-TM1;

[0025] Figure 6The results of SDS-PAGE analysis of SUMO-CBM-TM2 and GST-CBM-TM2 binding to cellulose; wherein, (a): SDS-PAGE analysis of SUMO-CBM-TM2 binding to cellulose, Lane M: protein marker; Lane 1: E. coli BL21 / pET-28a-SUMO 37℃ 0.1mM IPTG induced sample; Lane 2: E. coli BL21 / pSUMO-CBM-TM2 37℃ uninduced supernatant sample; Lane 3: E. coli BL21 / pSUMO-CBM-TM2 16℃ 0.05mM IPTG induced whole bacteria sample; Lane 4: E. coli BL21 / pSUMO-CBM-TM2 16℃ 0.05mM IPTG induced supernatant sample (before adsorption); Lane 5: E. coli BL21 / pSUMO-CBM-TM2 16℃ 0.05mM IPTG-induced supernatant sample (after adsorption); Lane 6: supernatant after desorption of cellulose-bound SUMO-CBM-TM2; (b): SDS-PAGE analysis of the binding ability of GST-CBM-TM2 to cellulose, Lane M: protein marker; Lane 1: E. coli BL21 / pGEX-4T-1 37℃ 0.1mM IPTG-induced sample; Lane 2: E. coli BL21 / pGST-CBM-TM2 37℃ uninduced supernatant sample; Lane 3: E. coli BL21 / pGST-CBM-TM2 16℃ 0.1mM IPTG-induced whole bacteria sample; Lane 4: E. coli BL21 / pGST-CBM-TM2 16℃ 0.1mM IPTG-induced supernatant sample (before adsorption); Lane 5: E. coli BL21 / pGST-CBM-TM2 16℃ 0.1mM IPTG-induced supernatant sample (after adsorption); Lane 6: supernatant after desorption of cellulose-bound GST-CBM-TM2;

[0026] Figure 7The results of SDS-PAGE analysis of the binding ability of CBM-MTT5 and SUMO-CBM-MTT5 to cellulose; wherein, (a): expression of CBM-MTT5 in E. coli BL21, Lane M: protein marker; Lane 1: supernatant sample of E. coli BL21 / pET-28a; Lane 2: whole bacteria induced by 16℃0.1mM IPTG of E. coli BL21 / pET28a-CBM-MTT5; Lane 3: supernatant induced by 16℃0.1mM IPTG of E. coli BL21 / pET28a-CBM-MTT5 (before adsorption); Lane 4: supernatant induced by 16℃0.1mM IPTG of E. coli BL21 / pET28a-CBM-MTT5 (after adsorption); (b): SDS-PAGE analysis of the binding ability of SUMO-CBM-MTT5 to cellulose, Lane M: protein marker; Lane Lane 1: E. coli BL21 / pET-28a-SUMO induced with 0.1 mM IPTG at 37°C; Lane 2: E. coli BL21 / pSUMO-CBM-MTT5 induced with 0.1 mM IPTG at 16°C; Lane 3: E. coli BL21 / pSUMO-CBM-MTT5 induced with 0.1 mM IPTG at 16°C (before adsorption); Lane 4: E. coli BL21 / pSUMO-CBM-MTT5 induced with 0.1 mM IPTG at 16°C (after adsorption); Lane 5: supernatant after desorption of cellulose-bound SUMO-CBM-MTT5;

[0027] Figure 8The results of SDS-PAGE analysis of SUMO-CBM-MTT5 binding efficiency to cellulose; (a): SDS-PAGE analysis of SUMO-CBM-MTT5 protein amount at different concentrations, Lane M: protein marker; Lane 1: supernatant induced by 0.1 mM IPTG at 37°C for E. coli BL21 / pET-28a-SUMO; Lane 2: supernatant without induction at 37°C for E. coli BL21 / pSUMO-CBM-MTT5; Lanes 3-7: supernatant samples of 0.4, 0.8, 1.2, 1.6, 2 mg / mL induced by 0.1 mM IPTG at 16°C for E. coli BL21 / pSUMO-CBM-MTT5 (before adsorption); (b): SDS-PAGE analysis of cellulose adsorption effect of SUMO-CBM-MTT5 at different concentrations, Lane M: protein marker; Lane 1: supernatant of 0.4, 0.8, 1.2, 1.6, 2 mg / mL for E. coli BL21 / pET-28a-SUMO 37℃ uninduced supernatant; Lane 2: supernatant induced by E. coli BL21 / pET-28a-SUMO at 37℃ and 0.1mM IPTG; Lane 3: uninduced supernatant of E. coli BL21 / pSUMO-CBM-MTT5 at 37℃; Lane 4-8: supernatant samples of E. coli BL21 / pSUMO-CBM-MTT5 induced by 16℃ and 0.4, 0.8, 1.2, 1.6, 2mg / mL (after adsorption); (c): SDS-PAGE analysis of cellulose desorption of SUMO-CBM-MTT5 at different concentrations, Lane M: protein marker; Lane 1: supernatant induced by E. coli BL21 / pET-28a-SUMO at 37℃ and 0.1mM IPTG; Lane 2: supernatant induced by E. coli BL21 / pSUMO-CBM-MTT5 at 37℃; Lane 3-7: supernatant after desorption of SUMO-CBM-MTT5 at cellulose-bound protein concentrations of 0.4, 0.8, 1.2, 1.6, and 2 mg / mL, (d): ImageJ grayscale scanning analysis of SUMO-CBM-MTT5 bands at protein concentrations of 1.2, 1.6, and 2 mg / mL after desorption, grayscale values ​​1-3 correspond to SUMO-CBM-MTT5 bands at protein concentrations of 1.2, 1.6, and 2 mg / mL;

[0028] Fig. 9The results of SDS-PAGE analysis of the storage stability of SUMO-CBM-MTT5 and cellulose-SUMO-CBM-MTT5; Lane M: protein marker; Lane 1-2: uninduced and induced supernatants of E. coli BL21 / pET-28a-SUMO at 37°C; Lane 3: uninduced supernatants of E. coli BL21 / pSUMO-CBM-MTT5 at 37°C; Lane 4: supernatants of E. coli BL21 / pSUMO-CBM-MTT5 induced at 16°C with 0.1 mM IPTG; Lane 5: supernatants of E. coli BL21 / pSUMO-CBM-MTT5 induced at 16°C with 0.1 mM IPTG and stored at 4°C for 7 days; Lane 6: supernatants of E. coli BL21 / pSUMO-CBM-MTT5 induced at 16°C with 0.1 mM IPTG and stored at -80°C for 7 days; Lane Lane 7: E. coli BL21 / pSUMO-CBM-MTT5 induced by 16℃ 0.1mM IPTG, supernatant cellulose adsorption, stored at 4℃ for 7 days; Lane 8: E. coli BL21 / pSUMO-CBM-MTT5 induced by 16℃ 0.1mM IPTG, supernatant cellulose adsorption, stored at 4℃ for 7 days;

[0029] Fig.10 The results of SDS-PAGE analysis of the binding stability of SUMO-CBM-MTT5 to cellulose, wherein Lane M: protein marker; Lane 1-2: uninduced and induced supernatants of E. coli BL21 / pET-28a-SUMO at 37°C; Lane 3: uninduced supernatant of E. coli BL21 / pSUMO-CBM-MTT5 at 37°C; Lane 4: supernatant induced by 0.1 mM IPTG at 16°C of E. coli BL21 / pSUMO-CBM-MTT5; Lane 5: supernatant induced by 0.1 mM IPTG at 16°C of E. coli BL21 / pSUMO-CBM-MTT5 (after adsorption); Lane 6: PBS buffer wash sample for preparing cellulose-SUMO-CBM-MTT5 adsorbent; Lane 7-8: 10 mM and 20 mM EDTA salt solution wash samples for preparing cellulose-SUMO-CBM-MTT5 adsorbent; Lane 9: Preparation of cellulose-SUMO-CBM-MTT5 adsorbent SUMO-CBM-MTT5 and cellulose desorption sample;

[0030] Fig.11The results of SDS-PAGE analysis of the binding of non-CBM component proteins to cellulose; wherein, (a): test of the binding of SUMO-tagged proteins to cellulose, Lane M: protein marker; Lane 1: uninduced supernatant of E. coli BL21 / pET-28a-SUMO at 37°C; Lanes 2-3: whole bacteria and supernatant of E. coli BL21 / pET-28a-SUMO induced at 37°C with 0.1mM IPTG (before adsorption); Lane 4: supernatant of E. coli BL21 / pET-28a-SUMO induced at 37°C with 0.1mM IPTG (after adsorption); Lane 5: supernatant after desorption of non-CBM component protein solution from cellulose; (b): test of the binding of GST-tagged proteins to cellulose, Lane M: protein marker; Lane 1: uninduced supernatant of E. coli BL21 / pGEX-6P-1 at 37°C; Lane Lane 2-3: whole bacteria and supernatant of E. coli BL21 / pGEX-6P-1 induced by 37℃ 0.1mM IPTG (before adsorption); Lane 4: supernatant of E. coli BL21 / pGEX-6P-1 induced by 37℃ 0.1mM IPTG (after adsorption); Lane 5: supernatant after desorption of non-CBM component protein solution and cellulose;

[0031] Fig.12 The results of scanning electron microscopy analysis of cellulose-SUMO-CBM-TM1 and cellulose-SUMO-CBM-TM2 are shown in Figure 1. (a) Cellulose; (b) Cellulose and SUMO-tagged protein; (c) Cellulose-SUMO-CBM-TM1; (d) Cellulose-SUMO-CBM-TM2;

[0032] Fig.13 This is the Fourier transform infrared spectrum analysis result of cellulose-GST-CBM-MTT1;

[0033] Fig.14 This is the Fourier transform infrared spectrum analysis result of cellulose-SUMO-CBM-MTT5;

[0034] Fig.15 Cellulose-GST-CBM-MTT1 reacts with heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption capacity analysis results; (a): cellulose-GST-CBM-MTT1 for heavy metal ions (Pb 2+ 、Cd 2+ ) removal rate; (b): cellulose-GST-CBM-MTT1 for heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption amount;

[0035] Fig.16 The cellulose-SUMO-CBM-TM1 reacts with heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption capacity analysis results; (a): cellulose-SUMO-CBM-TM1 for heavy metal ions (Pb 2+ 、Cd 2+ ) removal rate; (b): removal rate of heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption amount;

[0036] Fig.17 The cellulose-SUMO-CBM-TM2 reacts with heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption capacity analysis results, where (a) cellulose-SUMO-CBM-TM2 for heavy metal ions (Pb 2+ 、Cd 2+ ) removal rate; (b): removal rate of heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption amount;

[0037] Fig.18 The effect of cellulose-SUMO-CBM-MTT5 on heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption capacity analysis results, where (a) cellulose-SUMO-CBM-MTT5 for heavy metal ions (Pb 2+ 、Cd 2+ ) removal rate; (b): removal rate of heavy metal ions (Pb 2+ 、Cd 2+ ) adsorption amount;

[0038] Fig.19 Adsorption of Cd by cellulose-SUMO-CBM-TM1 2+ Elemental scanning analysis and semi-quantitative test results; (a): Elemental scanning analysis of cellulose-SUMO-CBM-TM1; (b): Cd adsorption on cellulose-SUMO-CBM-TM1 2+ Elemental scanning analysis; (c): semi-quantitative elemental analysis of cellulose-SUMO-CBM-TM1; (d): adsorption of Cd by cellulose-SUMO-CBM-TM1 2+ Semi-quantitative test of elements;

[0039] Fig. 20 Adsorption of Cd by cellulose-SUMO-CBM-TM22+ Elemental scanning analysis and semi-quantitative test results, where (a) is the elemental scanning analysis of cellulose-SUMO-CBM-TM2; (b) is the adsorption of Cd on cellulose-SUMO-CBM-TM2. 2+ Elemental scanning analysis; (c): semi-quantitative elemental analysis of cellulose-SUMO-CBM-TM2; (d): adsorption of Cd by cellulose-SUMO-CBM-TM2 2+ Semi-quantitative test of elements;

[0040] Fig.21 Pb adsorption for cellulose-SUMO-CBM-MTT5 2+ Elemental scanning analysis and semi-quantitative test results; (a): Elemental scanning analysis of cellulose-SUMO-CBM-MTT5; (b): Pb adsorption of cellulose-SUMO-CBM-MTT5 2+ Elemental scanning analysis; (c): cellulose-SUMO-CBM-MTT5 elemental semi-quantitative test; (d): cellulose-SUMO-CBM-MTT5 adsorption of Pb 2+ Semi-quantitative test of elements. DETAILED DESCRIPTION

[0041] The present invention provides a fusion protein, comprising a CBM and a metallothionein connected sequentially from the C-terminus to the N-terminus; the metallothionein comprises MTT1, MTT5, TM1 or TM2; the amino acid sequence of the MTT1 is shown in SEQ ID NO.1; the amino acid sequence of the MTT5 is shown in SEQ ID NO.2; the amino acid sequence of the TM1 is shown in SEQ ID NO.3; the amino acid sequence of the TM2 is shown in SEQ ID NO.4; and the amino acid sequence of the CBM is shown in SEQ ID NO.5.

[0042] In the present invention, the amino acid sequence of MTT1 is shown in SEQ ID NO.1, specifically: MDKVNSCCCGVNAKPCCTDPNSGCCCVSKTDNCCKSDTKECCTGTGEGCKCVNCKCCKPQANCC CGVNAKPCCFDPNSGCCCVSKTNNCCKSDTKECCTGTGEGCKCTSCQCCKPVQQGCCCGDKAKA CCTDPNSGCCCSNKANKCCDATSKQECQTCQCCK.

[0043] In the present invention, the amino acid sequence of MTT5 is shown in SEQ ID NO.2, specifically: MDKISGESTKICSKTEEKWCCCPSETQNCCNSDDKQCCVGSGEGCIYVCCKCCKVQAECKCGPNAKYCCIDPNTGNCCVCKTKFCSKSDSKECCPGGSC.

[0044] In the present invention, the amino acid sequence of TM1 is shown in SEQ ID NO.3, specifically: MDKVNSCCCGVNAKPCCTDPNSGCCCVSKTDNCCKSDTKECCTGTGEGCKCVNCKCCK.

[0045] In the present invention, the amino acid sequence of TM2 is shown in SEQ ID NO.4, specifically: MDKVNSCCCGVNAKPCCTDPNSGCCCVSKTDNCCKSDTKECCTGTGEGCKCVNCKCCKPQANCC CGVNAKPCCFDPNSGCCCVSKTNNCCKSDTKECCTGTGEGCKCTSCQCCK.

[0046] In the present invention, the amino acid sequence of the CBM is shown in SEQ ID NO.5, specifically: MDLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGS YNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSA SQFVEWDQVTAYLNGVLVWGKEPG.

[0047] In the present invention, the amino acid sequences of metallothionein MTT1 and MTT5 are obtained from the Tetrahymena genome database (http: / / www.ciliate.org). In the present invention, the MTT1 sequence is truncated, and only the first sequence module of MTT1 is retained as TM1, and the first and second sequence modules are retained as TM2.

[0048] In the present invention, the CBM and metallothionein are preferably connected by a flexible linker; the amino acid sequence of the flexible linker is shown in SEQ ID NO.6, specifically: GSAGSA. In the present invention, the use of a flexible linker can ensure that the metallothionein (MTT1, MTT5) and carbohydrate CBM components in the fusion protein can be correctly folded and effectively function.

[0049] In one embodiment of the present invention, the fusion protein comprises CBM and MTT1 connected sequentially from the C-terminus to the N-terminus, named CBM-MTT1, and the amino acid sequence is as shown in SEQ ID As shown in NO.7, MDLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGSYNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFVEWDQVTAYLNGVLVWGKEPGGSAGSAMDKV NSCCCGVNAKPCCTDPNSGCCCVSKTDNCCKSDTKECCTGTGEGCKCVNCKCCKPQANCCCGVNAKPCCFDPNSGCCCVSKTNNCCKSDTKECCTGTGEGCKCTSCQCCKPVQQGCCCGDKAKACCTDPNSGCCCSNKANKCCDATSKQECQTCQCCK, with a predicted size of approximately 34.57kDa.

[0050] In another embodiment of the present invention, the fusion protein includes CBM and MTT5 connected sequentially from the C-terminus to the N-terminus, named CBM-MTT5, and the amino acid sequence is shown in SEQ ID NO.8, specifically: MDLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGSYNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFVEWDQVTAYLNGVLVWGKEPGGSAGSAMDKISGESTKICSKTEEKWCCCPSETQNCCNSDDKQCCVGSGEGCIYVCCKCCKVQAECKCGPNAKYCCIDPNTGNCCVCKTKFCSKSDSKECCPGGSC, with a predicted size of approximately 28.41 kDa.

[0051] In another embodiment of the present invention, the fusion protein includes CBM and TM1 connected sequentially from the C-terminus to the N-terminus, named CBM-TM1, and the amino acid sequence is shown in SEQ ID NO.9, specifically: MDLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGSYNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFVEWDQVTAYLNGVLVWGKEPGGSAGSAMDKVNSCCCGVNAKPCCTDPNSGCCCVSKTDNCCKSDTKECCTGTGEGCKCVNCKCCK.

[0052] In another embodiment of the present invention, the fusion protein includes CBM and TM2 connected sequentially from the C-terminus to the N-terminus, named CBM-TM2, and the amino acid sequence is shown in SEQ ID NO.10, specifically: MDLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGSYNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFVEWDQVTAYLNGVLVWGKEPGGSAGSAMDKVNSCCCGVNAKPCCTDPNSGCCCVSKTDNCCKSDTKECCTGTGEGCKCVNCKCCKPQANCCCGVNAKPCCFDPNSGCCCVSKTNNCCKSDTKECCTGTGEGCKCTSCQCCK.

[0053] In the present invention, the C-terminus of the CBM is preferably modified with a tag; the tag preferably includes SUMO or GST. In the present invention, the tag helps to screen and obtain a sufficient amount of protein components.

[0054] The present invention also provides a nucleic acid molecule encoding the fusion protein described in the above scheme.

[0055] In the present invention, the nucleotide sequence of the nucleic acid molecule CBM-MTT1 encoding the CBM-MTT1 is shown in SEQ ID NO.11, specifically:

[0056] atggacctgaaagtggagttctacaactccaatccgtccgacaccaccaactccatcaatccgcagttcaaagttaccaacaccggtagcagcgcgatcgatctgtctaaactgacgctgcgttactactacactgtggatggtcagaaagaccagaccttctggtgcgatcacgcggcaatcatcggttccaacggtagctacaacggcatcacctccaacgtgaaaggcaccttcgttaagatgtcttcttctaccaacaacgctgatacctacctggaaatcagcttcaccggcggtactttggaaccaggcgcgcacgttcagatccagggtcgctttgcgaagaacgactggagcaactacacgcagagcaacgactactctttcaaatccgcttctcagttcgtggaatgggaccaggtgactgcctacctgaacggtgttctggtgtggggcaaagaaccgggcggttctgcaggctccgcaatggataaagttaactcttgctgctgtggtgtgaacgctaaaccgtgctgcactgacccgaactctggttgctgttgtgtgtccaagaccgataactgctgcaaatccgacaccaaagaatgttgcaccggcaccggtgaaggttgcaaatgcgtgaactgtaaatgctgcaaaccgcaggctaactgctgttgcggcgttaacgcaaagccgtgctgcttcgatccaaactccggttgttgttgcgttagcaagaccaacaactgctgtaaatccgataccaaagaatgctgtacgggcaccggcgaaggttgtaaatgcaccagctgtcagtgctgtaaaccagtacagcagggctgctgctgcggcgacaaagctaaagcatgctgcaccgacccgaacagcggctgctgctgctccaacaaagcgaacaaatgctgcgacgctacctctaaacaagaatgccagacttgccagtgctgcaag, The fragment size is 975bp.

[0057] In the present invention, the nucleotide sequence of the nucleic acid molecule CBM-MTT5 encoding the CBM-MTT5 is shown in SEQ ID NO.12, specifically:

[0058] , the fragment size is 786bp.

[0059] In the present invention, the nucleotide sequence of the nucleic acid molecule CBM-TM1 encoding the CBM-TM1 is shown in SEQ ID NO.13, specifically:

[0060] atggacctgaaagtggagttctacaactccaatccgtccgacaccaccaactccatcaatccgcagttcaaagttaccaacaccggtagcagcgcgatcgatctgtctaaactgacgctgcgttactactacactgtggatggtcagaaagaccagaccttctggtgcgatcacgcggcaatcatcggttccaacggtagctacaacggcatcacctccaacgtgaaaggcaccttcgttaagatgtcttcttctaccaacaacgctgatacctacctggaaatcagcttcaccggcggtactttggaaccaggcgcgcacgttcagatccagggtcgctttgcgaagaacgactggagcaactacacgcagagcaacgactactctttcaaatccgcttctcagttcgtggaatgggaccaggtgactgcctacctgaacggtgttctggtgtggggcaaagaaccgggcggttctgcaggctccgcaatggataaagttaactcttgctgctgtggtgtgaacgctaaaccgtgctgcactgacccgaactctggttgctgttgtgtgtccaagaccgataactgctgcaaatccgacaccaaagaatgttgcaccggcaccggtgaaggttgcaaatgcgtgaactgtaaatgctgcaaa。

[0061] In the present invention, the nucleotide sequence of the nucleic acid molecule CBM-TM2 encoding the CBM-TM2 is as shown in SEQ ID NO.14, specifically:

[0062] .

[0063] The present invention also provides a recombinant vector into which the nucleic acid molecule described in the above scheme is inserted. The present invention has no special restrictions on the construction method of the recombinant vector, and conventional methods in the art can be used.

[0064] In the present invention, the backbone plasmid of the vector preferably includes pET28a, pET-28a-SUMO, pGEX-6P-1 or pGEX-4T-1, and more preferably includes pET-28a-SUMO, pGEX-6P-1 or pGEX-4T-1. In the present invention, when the fusion protein is CBM-MTT1, the backbone plasmid of the recombinant vector preferably includes pGEX-6P-1; when the fusion protein is CBM-MTT5, the backbone plasmid of the recombinant vector preferably includes pET-28a-SUMO; when the fusion protein is CBM-TM1 and / or CBM-TM2, the backbone plasmid of the recombinant vector preferably includes pET-28a-SUMO and / or pGEX-4T-1. In the present invention, the recombinant plasmid is preferably constructed by Qingke Biotechnology Co., Ltd.

[0065] In one embodiment of the present invention, the backbone plasmid of the recombinant vector is pET28a, into which the coding gene of CBM-MTT1 is inserted, and the insertion sites are restriction endonuclease BamHI and XhoⅠ restriction sites; the recombinant vector is named pET28a-CBM-MTT1.

[0066] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pET28a, into which the coding gene of CBM-MTT5 is inserted, and the insertion sites are restriction endonuclease BamHI and XhoⅠ restriction sites; the recombinant vector is named pET28a-CBM-MTT5.

[0067] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pET-28a-SUMO, into which the coding gene of CBM-MTT1 is inserted, and the insertion sites are restriction endonuclease BamHⅠ and XhoⅠ restriction sites; the recombinant vector is named pSUMO-CBM-MTT1.

[0068] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pET-28a-SUMO, into which the coding gene of CBM-MTT5 is inserted, and the insertion sites are restriction endonuclease BamHI and XhoⅠ restriction sites; the recombinant vector is named pSUMO-CBM-MTT5. In the present invention, the SUMO tag can promote the soluble expression of CBM-MTT5.

[0069] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pGEX-6P-1, into which the coding gene of CBM-MTT1 is inserted, and the insertion sites are restriction endonuclease BamHI and XhoⅠ restriction sites; the recombinant vector is named pGST-CBM-MTT1. The GST fusion tag can promote the soluble expression of CBM-MTT1.

[0070] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pET-28a-SUMO, into which the coding gene CBM-TM1 is inserted, and the insertion sites are restriction endonuclease BamHI and XhoⅠ restriction sites; the recombinant vector is named pSUMO-CBM-TM1. The SUMO fusion tag promotes the soluble expression of CBM-TM1.

[0071] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pET-28a-SUMO, into which the CBM-TM2 coding gene CBM-TM2 is inserted, and the insertion sites are restriction endonuclease BamHⅠ and XhoⅠ restriction sites; the recombinant vector is named pSUMO-CBM-TM2.

[0072] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pGEX-4T-1, into which the CBM-TM1 coding gene CBM-TM1 is inserted, and the insertion sites are restriction endonuclease BamHI and XhoⅠ restriction sites; the recombinant vector is named pGST-CBM-TM1. The GST fusion tag promotes the soluble expression of CBM-TM1

[0073] In another embodiment of the present invention, the backbone plasmid of the recombinant vector is pGEX-4T-1, into which the CBM-TM2 coding gene CBM-TM2 is inserted, and the insertion sites are restriction endonuclease BamHⅠ and XhoⅠ restriction sites; the recombinant vector is named pGST-CBM-TM2.

[0074] The present invention also provides a host cell comprising the recombinant vector described in the above scheme.

[0075] The present invention has no particular limitation on the method for constructing the host cell, and conventional methods in the art may be used.

[0076] In the present invention, the original cell of the host cell includes Escherichia coli; the Escherichia coli is preferably Escherichia coli BL21 (DE3).

[0077] The present invention also provides a method for preparing the fusion protein described in the above scheme, comprising the following steps: inducing and culturing the host cells described in the above scheme to obtain an induced culture product.

[0078] In the present invention, the induction agent for the induction culture preferably includes IPTG; the working concentration of the IPTG preferably includes 0-0.2 mMol / mL, more preferably 0, 0.05 mMol / mL, 0.1 mMol / mL or 0.2 mMol / mL; the temperature of the induction culture is preferably 16-37°C, more preferably 16°C, 24°C or 37°C. In the present invention, when the temperature of the induction culture is 16°C, the time of the induction culture is preferably 12-16h; when the temperature of the induction culture is 37°C, the time of the induction culture is preferably 4h; when the temperature of the induction culture is 24°C, the time of the induction culture is preferably 12h.

[0079] In one embodiment of the present invention, the host cell is obtained by transferring the recombinant plasmid pET28a-CBM-MTT1 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0, 0.05, 0.1 or 0.2 mM, and the temperature of the induction culture is 16 or 37° C. In this embodiment, the CBM-MTT1 protein is successfully expressed, the expression amount of the target protein CBM-MTT1 is low, and the expressed fusion protein is mostly present in the form of inclusion bodies.

[0080] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant plasmid pET28a-CBM-MTT5 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0, 0.05, 0.1 or 0.2 mM, and the temperature of the induction culture is 16 or 37° C. In this embodiment, CBM-MTT5 protein is successfully expressed, and more soluble protein is expressed at 16° C.

[0081] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant plasmid pSUMO-CBM-MTT1 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0, and the temperature of the induction culture is 24° C. In this embodiment, the fusion protein SUMO-CBM-MTT1 is successfully expressed.

[0082] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant plasmid pSUMO-CBM-MTT5 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0.1 mM, the induction culture temperature is 16° C., and the induction culture time is 16 h. In this embodiment, the fusion protein SUMO-CBM-MTT5 is successfully expressed.

[0083] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant plasmid pGST-CBM-MTT1 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0.2 mM, the temperature of the induction culture is 37° C., and the time of the induction culture is 4 hours. In this embodiment, the fusion protein GST-CBM-MTT1 is successfully expressed, and more soluble protein can be obtained under the above conditions.

[0084] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant plasmid pSUMO-CBM-TM1 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0.05, 0.1 or 0.2 mM, preferably 0.1 mM; the temperature of the induction culture is 16 or 37°C, preferably 16°C; when the temperature of the induction culture is 37°C, the time of the induction culture is 4h; when the temperature of the induction culture is 16°C, the time of the induction culture is 16h. In this embodiment, the fusion protein SUMO-CBM-TM1 is successfully expressed, and more soluble protein can be obtained by induction at 16°C and 0.1mM IPTG for 16h.

[0085] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant plasmid pSUMO-CBM-TM2 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0.05, 0.1 or 0.2mM, preferably 0.05 or 0.1mM; the temperature of the induction culture is 16 or 37°C, preferably 16°C; when the temperature of the induction culture is 37°C, the time of the induction culture is 4h; when the temperature of the induction culture is 16°C, the time of the induction culture is 16h. In this embodiment, the fusion protein SUMO-CBM-TM2 is successfully expressed, and more soluble protein can be obtained at 16°C, 0.05 or 0.1mM IPTG induction for 16h, and the amount of soluble protein under 0.05mM IPTG induction is slightly higher than that under 0.1mM IPTG induction.

[0086] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant plasmid pGST-CBM-TM1 into Escherichia coli BL21 (DE3) cells, the working concentration of IPTG is 0.05, 0.1 or 0.2 mM, preferably 0.1 mM; the temperature of the induction culture is 16 or 37°C, preferably 37°C; when the temperature of the induction culture is 37°C, the time of the induction culture is 4h; when the temperature of the induction culture is 16°C, the time of the induction culture is 16h. In this embodiment, the fusion protein GST-CBM-TM1 is successfully expressed, and more soluble protein can be obtained by induction at 37°C and 0.1mM IPTG for 4h.

[0087] In another embodiment of the present invention, the host cell is obtained by transferring the recombinant plasmid pGST-CBM-TM2 into Escherichia coli BL21 (DE3) cells, the working concentration of the IPTG is 0.05, 0.1 or 0.2 mM, preferably 0.1 or 0.2 mM; the temperature of the induction culture is 16 or 37 ° C, preferably 16 ° C; when the temperature of the induction culture is 37 ° C, the time of the induction culture is 4 h; when the temperature of the induction culture is 16 ° C, the time of the induction culture is 16 h. In this embodiment, the fusion protein GST-CBM-TM2 is successfully expressed, and more soluble protein can be obtained at 16 ° C, 0.1 or 0.2 mM IPTG induction for 16 h, and the amount of soluble protein under 0.2 mM IPTG induction is slightly higher than that under 0.1 mM IPTG induction.

[0088] The present invention also provides a composite heavy metal adsorbent, which uses cellulose as a matrix and adsorbs the fusion protein described in the above scheme.

[0089] The main functional component in the composite heavy metal adsorbent of the present invention is the fusion protein, and the metallothionein MTT1, MTT5 and TM1, TM2 components in the fusion protein play a key role in the adsorption of heavy metal ions.

[0090] In the present invention, the cellulose material is the key matrix for binding and immobilizing the fusion protein, and the fusion protein is an indispensable component for modifying the cellulose.

[0091] The present invention also provides a method for preparing the composite heavy metal adsorbent described in the above scheme, comprising the following steps: mixing cellulose and a solution containing the fusion protein described in the above scheme, and adsorbing the mixture to obtain the composite heavy metal adsorbent.

[0092] In the present invention, cellulose is combined with protein to prepare an effective composite heavy metal adsorbent for adsorbing heavy metal ions, and these processes are all carried out in an aqueous solution environment.

[0093] In the present invention, the cellulose preferably comprises filter paper; the filter paper preferably comprises Whatman No. 1 filter paper; in the specific implementation process, the Whatman No. 1 filter paper is cut into small pieces with a diameter of about 0.6 cm to increase the contact surface area between the adsorbent and the heavy metal ions. Whatman No. 1 filter paper is resistant to immersion and has good reusability, and can maintain its shape without the application of mechanical force. In the present invention, the cellulose can be used as an adsorption carrier for the preparation of a cellulose-based composite heavy metal adsorbent.

[0094] In the present invention, the concentration of the fusion protein in the solution containing the fusion protein is preferably 0.4-2 mg / mL. In the specific implementation of the present invention, the concentration of the fusion protein in the solution containing the fusion protein is preferably 0.4, 0.8, 1.2, 1.6, or 2 mg / mL. The ratio of the mass of the cellulose to the volume of the solution containing the fusion protein is preferably 0.1 g / 1-4 mL. In the specific implementation of the present invention, the ratio of the mass of the cellulose to the volume of the solution containing the fusion protein is preferably 0.1 g / 1 mL, 0.1 g / 2 mL, 0.1 g / 3 mL, or 0.1 g / 4 mL.

[0095] In one embodiment of the present invention, the metallothionein in the fusion protein is MTT1, and the ratio of the mass of the cellulose to the volume of the solution containing the fusion protein is 0.1 g / 4 mL.

[0096] In another embodiment of the present invention, the metallothionein in the fusion protein is MTT5, and the ratio of the mass of the cellulose to the volume of the solution containing the fusion protein is 0.1 g / 1 mL.

[0097] In another embodiment of the present invention, the metallothionein in the fusion protein is TM1, and the ratio of the mass of the cellulose to the volume of the solution containing the fusion protein is 0.1 g / 1 mL.

[0098] In another embodiment of the present invention, the metallothionein in the fusion protein is TM2, and the ratio of the mass of the cellulose to the volume of the solution containing the fusion protein is 0.1 g / 2 mL.

[0099] In the present invention, the mixed system preferably also includes PBS buffer to ensure the stability of the protein; based on 0.05 g of cellulose, the added amount of PBS buffer is preferably 0.5 mL.

[0100] In the present invention, the adsorption conditions are preferably: 25° C., 100 rpm, adsorption for 2 h.

[0101] The present invention also provides the use of the fusion protein, the nucleic acid molecule, the recombinant vector, the host cell, the composite heavy metal adsorbent or the composite heavy metal adsorbent prepared by the preparation method described in the above scheme in at least one of the following: 1) heavy metal adsorption; 2) heavy metal enrichment; 3) heavy metal pollution control.

[0102] In the present invention, the heavy metal preferably includes Pb 2+ and / or Cd 2+ .

[0103] The present invention also provides a method for heavy metal adsorption or heavy metal enrichment, comprising the following steps:

[0104] The composite heavy metal adsorbent described in the above scheme or the composite heavy metal adsorbent prepared by the preparation method is mixed with an article containing heavy metals for adsorption.

[0105] In the present invention, the article containing heavy metals is preferably a solution containing heavy metals; the concentration of heavy metals in the solution containing heavy metals is preferably ≤40 mg / L; the pH value of the article containing heavy metals is preferably 6.8-7.2, which is conducive to the function of the protein component.

[0106] The present invention also provides a method for treating heavy metal pollution, comprising the following steps:

[0107] The composite heavy metal adsorbent described in the above scheme or the composite heavy metal adsorbent prepared by the preparation method is applied to the soil or water body to be treated for adsorption.

[0108] In order to further illustrate the present invention, the preparation and application of the composite heavy metal adsorbent provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0109] Test Example 1 Performance Test of Cellulose Materials

[0110] The cellulose material selected is Whatman No. 1 filter paper, which is cut into small pieces with a diameter of about 0.6 cm. In the process of preparing the adsorbent and removing heavy metal ions in the water environment, it is necessary to soak in liquid, so the cellulose material (filter paper) must be tested for its soakability and reusability in water. First, the cellulose material is repeatedly soaked and dried in a cycle of 12 hours, and repeated for 3 cycles (immersion + drying is one cycle) for a total of 72 hours; second, the cellulose material is dried after continuous soaking for 72 hours (0.1g cellulose is soaked in 20mL of clean water), and its dry weight is weighed after the cellulose is completely dried. The results are as follows: After repeated soaking and drying, it was found that this type of cellulose material is resistant to soaking and has good reusability, see Figure 1 (a) in; after continuous immersion and drying for 24 hours, the sample was weighed and found to be unable to maintain its shape under mechanical force, see Figure 1 (b) in the figure, and the dry weight remains unchanged (0.1 g) after drying.

[0111] Example 1 Preparation of Several Fusion Protein Composite Heavy Metal Adsorbents

[0112] Preparation flow chart of composite heavy metal adsorbent Figure 2 .

[0113] 1. Preparation of composite heavy metal adsorbents cellulose-SUMO-CBM-MTT1 and cellulose-GST-CBM-MTT1

[0114] The recombinant bacteria BL21 / pSUMO-CBM-MTT1 was constructed and induced to express, and the supernatant of the induced expression product was obtained to obtain the supernatant of the fusion protein SUMO-CBM-MTT1.

[0115] The recombinant bacteria BL21 / pGST-CBM-MTT1 was constructed and induced to express, and the supernatant of the induced expression product was obtained to obtain the supernatant of the fusion protein GST-CBM-MTT1.

[0116] The supernatants of the obtained fusion proteins SUMO-CBM-MTT1 and GST-CBM-MTT1 were used as fusion protein solutions (concentration of 1.55 mg / mL); 0.1 g of cellulose was added to 1 mL of the fusion protein solution, and adsorbed at 25°C and 100 rpm for 2 hours. When the timing was over, the supernatant after adsorption was taken and added to 5× protein loading buffer for sample preparation. The supernatant after adsorption was discarded, washed twice with 1 mL of deionized water, and then 1 mL of PBS buffer was added. 0.01 g of the adsorbed product cellulose-SUMO-CBM-MTT1 or GST-CBM-MTT1 was taken and added to PBS buffer and 5× protein loading buffer according to the final volume of 0.1 mL, and heated at 100°C for 10 minutes for desorption. The effect of cellulose adsorption of fusion protein was analyzed by 12% SDS-PAGE gel electrophoresis.

[0117] The results showed that the band of SUMO-CBM-MTT1 did not change significantly before and after adsorption, and there was no obvious band after desorption, see Figure 3 (a) in Figure 2. The band of GST-CBM-MTT1 disappeared compared with the control, see Figure 3 (b) in .

[0118] Compare the expression levels of SUMO-CBM-MTT1 and GST-CBM-MTT1 in the supernatant and their adsorption capacity to cellulose, and preliminarily select GST-CBM-MTT1 for the preparation of adsorbent. According to the adsorption of GST-CBM-MTT1 to cellulose, 0.1g cellulose was quantitatively added to different volumes of protein supernatant, and the selected fusion protein solution gradient was 1-4mL. The fusion protein solution was added to a 50mL centrifuge tube containing cellulose (the cellulose should be completely immersed in the fusion protein solution), and combined at 25℃ and 100rpm for 2h. Take the supernatant after adsorption for sample preparation, discard the remaining supernatant, wash twice with 1mL deionized water, add 1mL PBS buffer, take 0.01g adsorption product (based on cellulose dry weight) and add PBS buffer and 5× protein loading buffer according to the final volume of 0.1mL, and heat at 100℃ for 10min for sample preparation. The effect of cellulose adsorption of fusion protein and the amount of fusion protein adsorption were analyzed by 12% SDS-PAGE gel electrophoresis.

[0119] The results showed that the band of GST-CBM-MTT1 disappeared when the fusion protein solution was 1mL and 2mL, and gradually thickened when it increased to 3mL and 4mL. Correspondingly, after desorption, as the volume of the fusion protein solution increased, the band of GST-CBM-MTT1 also gradually thickened. When the fusion protein solution was close to 4mL, the band did not change significantly ( Figure 4 ), the binding amount of cellulose to GST-CBM-MTT1 reached saturation. In the adsorbent whose component was metallothionein MTT1, GST-CBM-MTT1 was selected to bind to cellulose and the composite heavy metal adsorbent cellulose-GST-CBM-MTT1 was prepared according to 0.1g cellulose / 4mL fusion protein solution.

[0120] 2. Preparation of composite heavy metal adsorbents cellulose-SUMO-CBM-TM1 and cellulose-GST-CBM-TM1

[0121] The recombinant bacteria BL21 / pSUMO-CBM-TM1 was constructed and induced to express, and the supernatant of the induced expression product was obtained to obtain the supernatant of the fusion protein SUMO-CBM-TM1.

[0122] The recombinant bacteria BL21 / pGST-CBM-TM1 was constructed and induced to express, and the supernatant of the induced expression product was obtained to obtain the supernatant of the fusion protein GST-CBM-TM1.

[0123] The supernatants of fusion proteins SUMO-CBM-TM1 and GST-CBM-TM1 were used as fusion protein solutions (concentrations of 2.19 mg / mL and 2.42 mg / mL), respectively. 0.5 mL of fusion protein solution and 0.5 mL of PBS buffer were added to 0.05 g of cellulose, and adsorbed at 25 ° C and 100 rpm for 2 hours. The supernatant after adsorption was added to 5× protein loading buffer for sample preparation. The remaining supernatant was discarded, and the adsorbed product was washed twice with 1 mL of deionized water, and then 0.05 mL of PBS buffer was added. 0.01 g of the binding product was added to PBS buffer and 5× protein loading buffer according to the final volume of 0.1 mL, and heated at 100 ° C for 10 minutes for desorption. The effect of cellulose adsorption of fusion proteins was analyzed by 12% SDS-PAGE gel electrophoresis and compared.

[0124] The results showed that the bands of SUMO-CBM-TM1 changed significantly before and after adsorption, and there were obvious bands after desorption, see Figure 5 (a) in the figure. The bands of GST-CBM-TM1 changed significantly before and after adsorption, and there were obvious bands after desorption. However, compared with SUMO-CBM-TM1 after desorption, GST-CBM-TM1 had more miscellaneous bands after desorption, see Figure 5 (b) In view of the expression of fusion protein SUMO-CBM-TM1 in the protein supernatant and its relatively small molecular weight, SUMO-CBM-TM1 was selected as the metallothionein TM1 component and combined with cellulose at a rate of 0.1 g cellulose / 1 mL fusion protein solution to prepare the composite heavy metal adsorbent cellulose-SUMO-CBM-TM1.

[0125] 3. Preparation of composite heavy metal adsorbents cellulose-SUMO-CBM-TM2 and cellulose-GST-CBM-TM2

[0126] The recombinant bacteria BL21 / pSUMO-CBM-TM2 was constructed and induced to express, and the supernatant of the induced expression product was obtained to obtain the supernatant of the fusion protein SUMO-CBM-TM2.

[0127] The recombinant bacteria BL21 / pGST-CBM-TM2 was constructed and induced to express, and the supernatant of the induced expression product was obtained to obtain the supernatant of the fusion protein GST-CBM-TM2.

[0128] The preparation of composite heavy metal adsorbents cellulose-SUMO-CBM-TM2 and cellulose-GST-CBM-TM2 refers to step 2.

[0129] The results showed that the bands of SUMO-CBM-TM2 changed significantly before and after adsorption, and there were obvious bands after desorption, see Figure 6 (a) in Figure 3. The bands of GST-CBM-TM2 before and after adsorption changed significantly. The bands were more obvious after desorption. However, compared with SUMO-CBM-TM2 after desorption, the protein bands of GST-CBM-TM2 after desorption were weaker and the miscellaneous bands were more obvious. Figure 6 (b) in the figure shows that SUMO-CBM-TM2 has a stronger ability to bind to cellulose. The content of fusion protein SUMO-CBM-TM2 and GST-CBM-TM2 in the protein supernatant is similar, but SUMO-CBM-TM2 has a smaller molecular weight. In order to contrast with SUMO-CBM-TM1, SUMO-CBM-TM2 was selected as the metallothionein TM2 component and combined with cellulose at 0.1 g cellulose / 2 mL fusion protein solution to prepare a composite heavy metal adsorbent cellulose-SUMO-CBM-TM2.

[0130] 4. Preparation of composite heavy metal adsorbents cellulose adsorbed CBM-MTT5 and cellulose-SUMO-CBM-MTT5

[0131] The recombinant bacteria BL21 / pCBM-MTT5 was constructed and induced to express, and the supernatant of the induced expression product was obtained to obtain the supernatant of the fusion protein CBM-MTT5.

[0132] The recombinant bacteria BL21 / pSUMO-CBM-MTT5 was constructed and induced to express, and the supernatant of the induced expression product was obtained to obtain the supernatant of the fusion protein SUMO-CBM-MTT5.

[0133] The supernatants of the obtained fusion proteins CBM-MTT5 and pSUMO-CBM-MTT5 were used as fusion protein solutions, 0.1 g cellulose was added to 1 mL fusion protein solution, and adsorbed at 25°C and 100 rpm for 2 hours. After adsorption, the supernatant was added to 5× protein loading buffer for sample preparation. The effect of cellulose adsorption of fusion proteins was analyzed by 12% SDS-PAGE gel electrophoresis.

[0134] The results showed that the CBM-MTT5 bands changed significantly before and after adsorption, indicating the binding ability of the untagged fusion protein to cellulose. Figure 7 (a) in .

[0135] The supernatant of the obtained fusion protein SUMO-CBM-MTT5 was used as the fusion protein solution (concentration of 2.06 mg / mL), 0.1 g of cellulose was added to 1 mL of the fusion protein solution, and adsorbed at 25°C and 100 rpm for 2 hours. The supernatant after adsorption was taken and added to 5× protein loading buffer for sample preparation. The remaining supernatant after adsorption was discarded, washed twice with 1 mL of deionized water, and then 1 mL of PBS buffer was added. 0.01 g of the adsorbed product cellulose-SUMO-CBM-MTT5 was added to PBS buffer and 5× protein loading buffer according to the final volume of 0.1 mL, and heated at 100°C for 10 minutes for desorption. The effect of cellulose adsorption of fusion protein was analyzed by 12% SDS-PAGE gel electrophoresis.

[0136] The results showed that the bands of SUMO-CBM-MTT5 changed significantly before and after adsorption, and there were obvious bands after desorption, see Figure 7 (b) in the figure. The SUMO tag promotes the expression of the fusion protein in the supernatant and has almost no effect on the binding ability to cellulose. Therefore, SUMO-CBM-MTT5 was initially selected as the protein component for the preparation of the adsorbent.

[0137] In view of the adsorption of SUMO-CBM-MTT5 to cellulose, 0.1g cellulose was quantitatively added to the same volume of protein supernatant with different concentrations. The selected fusion protein solution gradient ratio was 0.2, 0.4, 0.6, 0.8, 1 (concentrations were 0.4, 0.8, 1.2, 1.6, 2 mg / mL). 1mL of protein dilution was added to a 2mL centrifuge tube weighing cellulose, and combined at 25°C and 100rpm for 2h. The supernatant after adsorption was taken for sample preparation, and the remaining supernatant was discarded, washed twice with 1mL deionized water, and then 1mL PBS buffer was added. 0.01g of adsorption product (based on dry weight) was added to PBS buffer and 5× protein loading buffer according to the final volume of 0.1mL, and heated at 100°C for 10min for desorption. The effect of cellulose adsorption fusion protein and the grayscale detection of protein adsorption were analyzed by 12% SDS-PAGE gel electrophoresis. At the same time, a fusion protein solution dilution ratio of 0.4 was selected to test the stability of the prepared adsorbent. After adsorption at 25°C and 100rpm for 2h, it was placed at 4°C for 7d. Similarly, the fusion protein solution was placed at 4°C and -80°C for 7d, and the supernatant was taken for sample preparation. 12% SDS-PAGE gel electrophoresis was used to analyze the protein stability and the stability of the prepared adsorbent. The binding stability of SUMO-CBM-MTT5 with cellulose was also tested. After obtaining the adsorbent, it was washed twice with deionized water, and then washed with PBS buffer and 10mM and 20mM EDTA salt solutions in turn, and the washing solution was collected for 12% SDS-PAGE gel electrophoresis analysis.

[0138] The results showed that the band changes of SUMO-CBM-MTT5 before and after adsorption at different protein concentrations were obvious and gradient-type. After desorption, it was found that when the protein concentration was 1.2 mg / mL (including higher concentrations), no obvious changes in the band were observed. The grayscale detection and analysis of the protein bands determined that when the protein concentration reached 1.2 mg / mL, the binding amount of SUMO-CBM-MTT5 to cellulose reached a saturated state, see Figure 8 Therefore, among the adsorbents whose component was MTT5, SUMO-CBM-MTT5 was selected to combine with cellulose and the composite heavy metal adsorbent cellulose-SUMO-CBM-MTT5 was prepared at 0.1 g cellulose / 1 mL fusion protein solution (protein concentration: 1.2 mg / mL).

[0139] The storage stability and binding stability of the initially prepared cellulose-SUMO-CBM-MTT5 adsorbent were verified. The results showed that the SUMO-CBM-MTT5 protein could be stably stored for 7 days at 4°C and -80°C without obvious degradation. The adsorbent could also be stably stored for 7 days at 4°C. Fig. 9); There were no obvious bands in the PBS and EDTA washing samples, indicating that PBS and EDTA had no obvious elution of SUMO-CBM-MTT5 bound to cellulose, indicating that the binding of SUMO-CBM-MTT5 to cellulose was relatively stable ( Fig.10 ).

[0140] Experimental Example 2: Binding test of non-CBM component proteins SUMO and GST-tagged proteins to cellulose

[0141] In order to further test whether the binding of CBM component proteins to cellulose is specific, non-CBM component proteins SUMO and GST-tagged proteins were selected as controls for comparison with the preparation of the above-mentioned composite heavy metal adsorbent. The obtained SUMO-tagged protein supernatant was added with 1 mL of fusion protein solution according to 0.1 g of cellulose, and oscillated at 25°C and 100 rpm for 2 h; while the GST-tagged protein supernatant was added with 0.5 mL of fusion protein solution and 0.5 mL of PBS buffer according to 0.05 g of cellulose, and oscillated at 25°C and 100 rpm for 2 h. The supernatant was taken for sample preparation, and the rest of the supernatant was discarded, washed twice with 1 mL of deionized water, and then 1 mL of PBS buffer was added. 0.01 g of adsorption product (based on dry weight) was added to PBS buffer and 5× protein loading buffer according to the final volume of 0.1 mL, and heated at 100°C for 10 min for sample preparation. 12% SDS-PAGE gel electrophoresis was used for gel running and identification.

[0142] The results showed that there was no significant change in the overall protein bands before and after adsorption, and there were no obvious bands or bands indicating adsorption after desorption ( Fig.11 ). This indicates that non-CBM component proteins are difficult to bind to cellulose or cannot effectively bind to cellulose, and the CBM component plays a major role in the binding of fusion protein to cellulose.

[0143] Test Example 3 Characterization of the Composite Heavy Metal Adsorbent Prepared in Example 1

[0144] The prepared composite heavy metal adsorbent was freeze-dried for 12 to 18 hours using a vacuum freeze dryer to obtain a freeze-dried product. Cellulose and non-CBM component protein cellulose binding products (freeze-dried products) soaked in PBS and freeze-dried were used as controls, and the morphology was characterized using a scanning electron microscope (SEM). Pure cellulose was used as a control, and the functional groups of cellulose-GST-CBM-MTT1, cellulose-SUMO-CBM-MTT5, cellulose-SUMO-CBM-TM1 and cellulose-SUMO-CBM-TM2 were detected using Fourier transform infrared spectroscopy (FTIR).

[0145] The morphology of cellulose-SUMO-CBM-TM1 and cellulose-SUMO-CBM-TM2 was characterized by scanning electron microscopy (SEM), and cellulose-GST-CBM-MTT1 and cellulose-SUMO-CBM-MTT5 were detected by Fourier transform infrared spectroscopy (FTIR). Compared with pure cellulose and non-CBM components, cellulose-SUMO-CBM-TM1 and cellulose-SUMO-CBM-TM2 have obvious changes in appearance. The pores on the cellulose surface are almost completely covered due to the binding of proteins ( Fig.12 ). The Fourier spectrum results showed that compared with pure fibers, cellulose-GST-CBM-MTT1 had a -1 and 1530cm -1 The protein characteristic peak (amide I band (1600-1700 cm -1 ), amide II band (1500~1600cm -1 ))( Fig.13 ), cellulose-SUMO-CBM-MTT5 at 1650 cm -1 and 1540cm -1 The protein characteristic peak appears at Fig.14 ). This shows the binding of protein to cellulose.

[0146] In summary, the successful preparation of composite heavy metal adsorbents cellulose-SUMO-CBM-TM1, cellulose-SUMO-CBM-TM2, cellulose-GST-CBM-MTT1 and cellulose-SUMO-CBM-MTT5, the changes in the morphology of the cellulose surface and the appearance of functional groups behind the cellulose binding protein also further confirmed the binding of cellulose and fusion protein, which is further evidence of the successful preparation of composite heavy metal adsorbents.

[0147] Example 2 The composite heavy metal adsorbent prepared in Example 1 is used for heavy metal ion Pb 2+ 、Cd 2+ Adsorption

[0148] Dilution of heavy metal ions (Pb 2+ 、Cd 2+ ) mother liquor, with an initial concentration of 40 mg / L. The prepared composite heavy metal adsorbents cellulose-GST-CBM-MTT1, cellulose-SUMO-CBM-TM1, cellulose-SUMO-CBM-TM2 and cellulose-SUMO-CBM-MTT5 were used for Pb 2+ 、Cd 2+ Adsorption experiment.

[0149] In order to reduce the influence of PBS buffer and attachments on the adsorption of heavy metal ions, the prepared adsorbent was washed twice with deionized water (the volume of deionized water corresponded to the volume of the fusion protein solution added when preparing the adsorbent), added to 40 mL of a heavy metal ion solution with an initial concentration of 40 mg / L (adjusted to pH ≈ 7 with NaOH), and adsorbed at 25 °C and 100 rpm for 3 h. After adsorption, the adsorbent was collected by centrifugation at 8,000 rpm for 10 min, and 5 mL of the solution was taken for determination of heavy metal ions (Pb 2+ 、Cd 2+ ) concentration. Each single ion adsorption experiment was performed in triplicate. The conical flask used was nitrated with 20% dilute nitric acid solution and reused.

[0150] Composite heavy metal adsorbent for Pb 2+ and Cd 2+ The removal rate (%) and adsorption capacity are calculated according to the following formula:

[0151] Removal rate: R (%) = (C0-C) / C0×100% (C0: initial concentration of heavy metal ions, C: concentration of heavy metal ions in the solution after adsorption)

[0152] Adsorption amount = (C0-C) × V / M DW (C0: initial concentration of heavy metal ions, C: concentration of heavy metal ions in the solution after adsorption, V: volume of heavy metal ion solution)

[0153] All experiments were repeated 3 times. SPSS Statistics 27.0 software was used for statistical analysis. Origin 8.0 was used for graphics. One-way ANOVA was used to analyze the differences between the control group and the experimental group (*p<0.05 indicates a significant difference, **p<0.01 indicates an extremely significant difference). Dry weight (DW) was used to calculate the corresponding parameters.

[0154] The results show:

[0155] Compared with pure cellulose, the composite heavy metal adsorbent cellulose-GST-CBM-MTT1 has significant differences in removal efficiency and adsorption capacity. 2+ The removal rate and adsorption capacity of Pb were 32.6% and 2.61 mg / g, respectively, while pure cellulose 2+ The removal rate and adsorption capacity of Cd were 18.7% and 1.49 mg / g respectively; the removal rate and adsorption capacity of Cd were 18.7% and 1.49 mg / g respectively for the composite heavy metal adsorbent cellulose-GST-CBM-MTT1. 2+ The removal rate and adsorption capacity of pure cellulose for Cd were 39.2% and 3.13 mg / g, respectively. 2+ The removal rate and adsorption capacity were 22.3% and 1.79 mg / g ( Fig.15 ).

[0156] The composite heavy metal adsorbent cellulose-SUMO-CBM-TM1 has a higher adsorption rate for Pb than pure fiber. 2+ 、Cd 2+ The ability of the composite heavy metal adsorbent cellulose-SUMO-CBM-TM1 to absorb Pb 2+ The removal rate and adsorption capacity of Cd were 59.3% and 4.75 mg / g respectively; the removal rate and adsorption capacity of Cd of composite heavy metal adsorbent cellulose-SUMO-CBM-TM1 were 59.3% and 4.75 mg / g respectively. 2+ The removal rate and adsorption capacity were 52.1% and 4.17 mg / g ( Fig.16 ).

[0157] The composite heavy metal adsorbent cellulose-SUMO-CBM-TM2 has a higher adsorption rate for Pb than pure fiber 2+ 、Cd 2+ There is a significant difference in the ability of Pb 2+ 、Cd 2+ The removal rates of Pb and 2+ 、Cd 2+ The adsorption amounts were 5.69 mg / g and 4.36 mg / g respectively. Fig.17 ).

[0158] Compared with pure fiber, the composite heavy metal adsorbent cellulose-SUMO-CBM-MTT5 adsorbs heavy metal ions, especially Pb 2+ The ability of Pb has been significantly improved. 2+ 、Cd 2+ The removal rates of Pb and 2+ 、Cd 2+ The adsorption amounts were 5.69 mg / g and 3.45 mg / g respectively. Fig.18 ).

[0159] In summary, compared with pure cellulose, it was found that each composite heavy metal adsorbent had a significant effect on the removal of heavy metal ions. The molecular weights of SUMO-CBM-TM2 and SUMO-CBM-MTT5 proteins were similar, and the amount of protein bound to cellulose was similar. Therefore, cellulose-SUMO-CBM-TM2 and cellulose-SUMO-CBM-MTT5 had a significant effect on the removal of heavy metal ions Pb. 2+ The adsorption amount of Cd 2+ The adsorption capacity of cellulose-SUMO-CBM-TM2 was slightly higher than that of cellulose-SUMO-CBM-MTT5. 2+ The ability is significantly stronger than Cd2+ .

[0160] Test Example 4: Adsorption of heavy metal ions Pb 2+ 、Cd 2+ Semi-quantitative test

[0161] Combined with the ICP-OES measurement results of the composite heavy metal adsorbent of Example 2, the composite heavy metal adsorbents cellulose-SUMO-CBM-TM1 and cellulose-SUMO-CBM-TM2 combined with Cd 2+ The product was freeze-dried using a vacuum freeze dryer and analyzed by SEM element scanning; the composite heavy metal adsorbent cellulose-SUMO-CBM-MTT5 adsorbed Pb 2+ After freeze-drying, the product was analyzed by SEM.

[0162] The results showed that cellulose-SUMO-CBM-TM1 and cellulose-SUMO-CBM-TM2 adsorbed Cd 2+ Afterwards, the element scanning analysis showed the presence of Cd, and the Cd content was high in the semi-quantitative detection ( Fig.19 , Fig. 20 ); Cellulose-SUMO-CBM-MTT5 adsorbs Pb 2+ After that, Pb was found in the elemental scanning analysis, and Pb accounted for a higher proportion in the semi-quantitative detection ( Fig.21 ). According to the results of SEM, it was confirmed that the composite heavy metal adsorbent has a strong affinity for heavy metal ions Pb 2+ and Cd 2+ The above results show that cellulose-SUMO-CBM-TM1 and cellulose-SUMO-CBM-TM2 can absorb Cd 2+ The semi-quantitative test also indirectly showed that the two 2+ The adsorption capacity of cellulose-SUMO-CBM-MTT5 was 2+ The elemental analysis and semi-quantitative detection of Pb 2+ combination.

[0163] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fusion protein, characterized in that It comprises a CBM and a metallothionein connected sequentially from the C-terminus to the N-terminus; the metallothionein comprises MTT1, MTT5, TM1 or TM2; the amino acid sequence of the MTT1 is shown in SEQ ID NO.1; the amino acid sequence of the MTT5 is shown in SEQ ID NO.2; the amino acid sequence of the TM1 is shown in SEQ ID NO.3; the amino acid sequence of the TM2 is shown in SEQ ID NO.4; and the amino acid sequence of the CBM is shown in SEQ ID NO.

5.

2. A nucleic acid molecule, characterized in that Encoding the fusion protein of claim 1.

3. A recombinant vector, characterized in that: The nucleic acid molecule according to claim 2 is inserted.

4. A host cell comprising the recombinant vector according to claim 4.

5. A composite heavy metal adsorbent, characterized in that: Cellulose is used as a matrix, and the fusion protein according to claim 1 is adsorbed.

6. The method for preparing the composite heavy metal adsorbent according to claim 5, characterized in that: The following steps are involved: The cellulose and the solution containing the fusion protein according to claim 1 are mixed and adsorbed to obtain a composite heavy metal adsorbent.

7. Use of the fusion protein of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3, the host cell of claim 4, the composite heavy metal adsorbent of claim 5 or the composite heavy metal adsorbent prepared by the preparation method of claim 6 in at least one of the following: 1) heavy metal adsorption; 2) heavy metal enrichment; 3) heavy metal pollution control.

8. The use according to claim 7, characterized in that: The heavy metals include Pb 2+ and / or Cd 2+ .

9. A method for heavy metal adsorption or heavy metal enrichment, characterized in that: The following steps are involved: The composite heavy metal adsorbent according to claim 5 or the composite heavy metal adsorbent prepared by the preparation method according to claim 6 is mixed with an article containing heavy metals for adsorption.

10. A method for controlling heavy metal pollution, characterized in that: The following steps are involved: The composite heavy metal adsorbent according to claim 5 or the composite heavy metal adsorbent prepared by the preparation method according to claim 6 is applied to the soil or water body to be treated for adsorption.

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