Polypeptide, composition and process for separating silica from a medium

Abstract

The present invention relates to the field of ore processing and flotation. More specifically, the present invention relates to the production of recombinant polypeptides, a composition comprising a silica-binding polypeptide and to a process for separating silica in a medium, where the process is preferably quartz flotation in iron ore.

Description

POLYPEPTIDE, COMPOSITION AND PROCESS FOR SEPARATING SILICA FROM A MEDIUM FIELD OF THE INVENTION

[0001] The present invention relates to the field of ore beneficiation, flotation, and mineralogy. More specifically, the present invention relates to the production of recombinant polypeptides, a composition comprising a silica-binding polypeptide, and a process for separating silica from a medium, wherein the process is preferably quartz flotation on iron ore. BACKGROUND OF THE INVENTION

[0002] To meet global demand in the iron and steel industry, exploration of deposits with complex mineralogy—that is, low iron content and large amounts of impurities, such as quartz, a crystalline form of silica—has been increasingly being conducted. However, to obtain a high-quality final product with a high iron concentration and low quartz content, a beneficiation stage is necessary, where impurities are removed through a flotation process. During flotation, synthetic chemical reagents are used to interact with and modify the mineral surface to facilitate the separation of the desired mineral species (Govender and Gericke, 2011; Oliveira, 2018; Rocha, 2021).

[0003] Among the flotation processes for concentrating iron ore containing quartz as an impurity, reverse cation flotation stands out. This process typically uses amines and ether amines to act as quartz collectors, while starch and its derivatives are added to promote the depression of iron oxides (Budemberg, 2016). Although the application of this set of reagents has been successful in industrial practice, efforts have been made to optimize process selectivity and minimize costs and environmental impacts. Amines used as collectors are expensive and have considerable toxicity levels. Therefore, the development of more selective collectors capable of reducing the need for depressant reagents could contribute to increased recovery and product quality in current processing plants (Silva et al., 2022).

[0004] In recent years, for example, it has been discussed that amidamines, a class of cationic surfactant compounds containing one or more amide groups as well as one or more amine groups, can be used as quartz collectors in the iron ore flotation process. According to some studies, these molecules can exhibit high selectivity for quartz, do not require the action of an iron depressant agent during flotation, and still promote high iron recovery (Budemberg, 2016; Rocha, 2021; Silva et al., 2021; Silva et al., 2022).

[0005] Alternatively, diatom microalgae produce a silica shell through the interaction of silica with specific proteins rich in lysine and arginine residues, which, when secreted by the cells, promote the formation of biogenic silica (Poulsen and Kröger, 2004). These polypeptides are called silica-binding proteins (SiBPs).

[0006] In this context, the present invention discloses a composition comprising a silica-binding polypeptide for use as a bioreagent in the beneficiation of silicate ores, such as in the reverse flotation of iron ore, presenting high selectivity for quartz and low environmental impact. More specifically, it proposes the use of silica-binding polypeptides (silaffins), referred to herein as SiBPs, derived from diatom algae, in the quartz flotation process. These molecules can also be produced in vivo, and the results of quartz microflotation with They are promising. A survey of the state of the art on SiBPs and their intended application in the project was conducted, but no documents have been found to date on this topic, much less on the use of these polypeptides in mineral flotation. Some documents that define the general state of the art regarding silica-binding polypeptides are listed below:

[0007] WO2009062713A1 refers to transgenic photosynthetic organisms with expanded absorption spectrum and nitrogen autotrophy, which are optionally salt-resistant and / or capable of separating silicate from the medium and / or have an increased hydrocarbon content. The document also reveals the use of these organisms in a wide variety of fields of study. Examples include their use in the extraction of biofuels and nitrogen-containing biomass, particularly to provide essential amino acids for humans and animals; for the sequestration of CO2 from the atmosphere; for the extraction of sugars, polyalkoxy compounds, paper, cardboard, corrugated board, cellulose, fiberboard, or cotton; fuels, propellants, or lubricants; and food for animals and humans; for improving poor soil; for processing wastewater; for the expression of useful proteins; for the breakdown of hydrophobic impurities in natural habitats; for mosquito control and emission protection.

[0008] Document US9920304B2 discloses a silica-synthesizing peptide and its uses. The silica-synthesizing peptide can polymerize silica from a silica precursor in an aqueous solution under conditions of normal temperature, normal pressure, and a near-neutral weak base. The silica-synthesizing peptide can form a self-assembled structure during silica synthesis, and therefore, the document cites several ways in which the claimed subject matter can be used—for example, it can be used as various biomaterials, such as a silica-based protein immobilizer, a biosensor, and a drug delivery system. The subject matter of the aforementioned North American document is further provided as a fusion protein including at least one peptide selected from the group consisting of amino acid sequences presented in SEQ ID NOs: 1 to 10 and a self-assembling protein, as well as a composition for synthesizing silica comprising said protein.

[0009] US20140363872 A1 provides materials and methods for producing non-naturally occurring, cell-surface-directed bioinorganic membranes for association with the cell surfaces of living cells. The methods comprise exposing a cell to an acidic biomineralization buffer environment for cell-mediated deposition of the biomineral membrane onto the cell surface. The methods also comprise attaching a peptide, having a net positive charge under the acidic conditions, to the cell surface to serve as a template for directing cell-mediated deposition of the biomineral membrane onto the cell surface.

[0010] US20140379094 A1 relates to a fusion protein comprising a fibrous protein domain and a mineralization domain. The claimed fusion is used to form an organic-inorganic composite. According to the disclosure, these organic-inorganic composites can be constructed from the nanoscale to the macroscale, depending on the size of the fibrous protein fusion domain used. In one embodiment of the invention in the aforementioned US document, the composites can also be loaded with other compounds (e.g., dyes, drugs, enzymes), depending on the purpose of the materials, to further enhance the desired function. This objective can be achieved during material assembly or during the mineralization step in material formation. Furthermore, paragraph 0072 notes that the process for mineralizing the coating depends on the mineralization domain that is part of the fusion protein of the invention. and, most importantly, that the mineralization domain of the Silafin protein is involved in silica precipitation.

[0011] US20080182971 A1 discloses methods for forming peptide derivatives using functional moieties and other peptide derivatives. Methods for using these peptide derivatives to form silicon-based composite materials and silicon-based composite materials thus formed are also provided. Silicon-based composite materials may have nanoscale characteristics and may exhibit characteristics derived from the functional moieties in the peptide derivatives. Specifically, peptides can be used as templates in the formation of silicon-based hybrid materials. The resulting silicon-based hybrid materials will have the functionality conferred by the functional group or groups in the peptides. It is noted in paragraph 0004 that silaffins represent a class of peptides found in diatoms that produce silica nanospheres.Paragraph 0026 mentions an embodiment of the invention being a peptide comprising a portion of the primary structure of the “Silp” protein that can be used as the claimed peptides.

[0012] Lechner, CC, Becker, CF: Silaffins in Silica Biomineralization and Biomimetic Silica Precipitation. Mar Drugs.2015d Aug 19;13(8):5297-333. doi: 10.3390 / md13085297 reports that in vitro silaffin-based peptides alone, with and without post-traditional modifications, can efficiently mediate biomimetic silica precipitation, leading to silica materials with different properties and encapsulated cargo molecules of a wide range of sizes. In this review article, the process of silica biomineralization in diatoms is summarized with a specific focus on silaffins and their in vitro silica precipitation properties. Applications in bio- and nanotechnology, as well as in diagnostics and therapeutics, are discussed. SUMMARY OF THE INVENTION

[0013] The present invention aims to provide a recombinant polypeptide, being SiBP2 or SiBP3, represented by SEQ ID NO: 1 and 2, respectively, in which said polypeptides are modified with the insertion of a 6-histidine (6His) tag in the amino-terminal portion. The SIPB2 polypeptide can further be modified to contain a flexible “GSGSGS” portion followed by 4 isoleucines (S4I) in the carboxy-terminal portion.

[0014] The aforementioned polypeptide, whether or not it carries the mentioned modifications, is used to separate silica efficiently and sustainably in a medium.

[0015] In particular, the separation of silica from a medium can be ore beneficiation, for example, iron ore. This beneficiation is preferably quartz flotation on iron ore.

[0016] The present invention further provides a composition comprising the polypeptide of SEQ ID NO: 1 and / or 2, and a suitable carrier, wherein the polypeptides are optionally modified by the insertion of a histidine tag at the amino-terminal portion. Optionally, the composition may contain an additive being alcohols, esters and oils, preferably ethanol, ethyl dodecanoate, essential oils such as lemongrass and / or eucalyptus.

[0017] In a third embodiment, the present invention provides a process for separating silica from a medium, wherein the process involves applying the composition as previously described. Optionally, to improve the separation of hematite and silica minerals, the depressant starch (approximately 10 mg / L, depending on the mineral system: percentage of each).

[0018] In particular, the composition is used in the flotation of quartz in iron ore. BRIEF DESCRIPTION OF THE FIGURES

[0019] Figure 1. SDS-PAGE analysis of the purification of recombinant SiBP2 polypeptides (a) and quartz microflotation tests using the 6His-SiBP2 polypeptide or its modified form 6His-SiBP24I, which contains an addition of 4 isoleucines at the carboxy-terminal region. (b) The 6His-SiBP2 polypeptide was purified by affinity liquid chromatography, dialyzed into 1x PBS to remove the imidazole eluent, and approximately 400 μg of SiBP2 in 200 μL of 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 adjusted to pH 7.4) were used in the microflotation tests with a final volume of 140 mL of deionized water. Addition of other components with SiBP2 or with the PBS control 1x (200 / 140,000 (v / v) were also tested to verify the quartz floatability. EtOH- ethanol 10 / 140,000 (v / v); 2EtD- 2 / 140,000 (v / v) of ethyl dodecanoate; 1CL- 1 / 140,000 (v / v) of lemongrass essential oil; 1Eu- 1 / 140,000 (v / v) of eucalyptus essential oil.Flotability was compared with a commercial reagent. * indicates significant difference (p < 0.05) in relation to the floatability of the commercial reagent. DETAILED DESCRIPTION OF THE INVENTION.

[0020] During the flotation of oxidized ores, exemplified here by the reverse flotation of iron ore, synthetic chemical reagents called collectors, such as amines and etheramines, are used to interact with and modify the mineral surface to facilitate the separation of contaminants such as quartz. These chemical reagents are expensive, can be polluting, and have low selectivity depending on the process conditions used.

[0021] The total or partial replacement of these chemical reagents by molecules of biological origin, maintaining selectivity for the compound The main interest is a demand in mineral flotation processes. In this context, the present invention relates to a bioreagent for processing ores containing silica, such as reverse flotation of iron ore, which presents high selectivity for quartz and has low environmental impact.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one skilled in the art to which the invention belongs. The terminology used in the description of the invention is intended to describe particular embodiments only and is not intended to limit the scope of the teachings. Unless otherwise indicated, all numbers expressing quantities, percentages, and proportions, and other numerical values ​​used in the specification and claims, should be understood as being modified, in all cases, by the term "about." Therefore, unless otherwise indicated, the numerical parameters shown in the specification and claims are approximations that may vary depending on the properties to be obtained.

[0023] Thus, according to a first aspect of the invention, recombinant polypeptides are provided, represented by SEQ ID NO: 1 and 2, in which said polypeptides are modified by the insertion of a histidine tail in the amino-terminal portion.

[0024] In one embodiment, the polypeptides may be further modified by the addition of four isoleucine residues at the carboxy-terminal portion.

[0025] In a second aspect of the invention, there is provided a composition comprising the polypeptides represented by SEQ ID NO: 1 or 2 and at least one suitable carrier.

[0026] In one embodiment, said polypeptides may be modified by inserting a histidine tag (6His) into the amino-terminal portion.

[0027] In another embodiment, the polypeptides present in the composition may be further modified by adding or deleting four isoleucines from the carboxy-terminal portion.

[0028] In another embodiment, the composition may also comprise an additive.

[0029] Preferably, the additive is an alcohol, ester or oil, more preferably a vegetable-derived additive, which may optionally be added to the flotation medium.

[0030] More preferably, the plant-derived additive may be ethanol 10 to 12 / 140,000 (v / v), ethyl dodecanoate 1 to 2 / 140,000 (v / v), essential oils such as lemongrass and / or eucalyptus essential oil 1 to 2 / 140,000 (v / v) or the depressant starch (10 mg / L).

[0031] More preferably, the plant additive is optionally applied in an amount in the range of about 1 part per 140,000 (v / v) in the flotation solution together with 1x PBS [200 to 400 parts per 140,000 (v / v)] containing 400 ^g of the polypeptide to a final volume of 140 mL (approximately 3 mg / L) in the flotation medium.

[0032] A concentration range of approximately 3 mg / L to 6 mg / L of polypeptides can be used to separate silica without additives or with the depressant starch (10 mg / L) to improve silica selectivity in mineral mixtures. However, in the modalities described in the previous paragraphs, the concentrations may vary and be adapted according to the percentage of each mineral in the flotation system.

[0033] In another embodiment, the present invention relates to a process for separating silica from a medium, comprising applying, to the medium from which the silica will be separated, a composition comprising the polypeptides represented by SEQ ID NO: 1 or 2 and a vehicle, optionally in which the polypeptides are modified with the addition of a tail of 6 histidines in the amino-terminal portion and / or modified with the deletion of 4 isoleucines in the carboxy-terminal portion, optionally in which the composition comprises an additive being an alcohol, ester or oil, preferably a vegetable additive selected from ethanol 10 to 12 / 140,000 (v / v), ethyl dodecanoate 1 to 2 / 140,000 (v / v), essential oils such as lemongrass and / or eucalyptus essential oil 1 to 2 / 140,000 (v / v).

[0034] Preferably, the process of separating silica from a medium is beneficiation of an ore.

[0035] Most preferably, the ore to be processed is iron ore.

[0036] Most preferably, the beneficiation process is quartz flotation.

[0037] In all modalities described in the previous paragraphs, the concentrations may vary and be adapted according to the percentage of each mineral in the flotation system, and should not be limited to the examples described here. EXAMPLES Example 1 - Cloning and heterologous expression of polypeptides that bind to silica (Silica-Binding Proteins – SiBPs)

[0038] In an attempt to produce a polypeptide-derived bioreagent, two genes encoding SiBP2 and SiBP3, whose GenBank accession numbers are XP_002286917 (SEQ ID NO: 1) and AAF16940.1 (SEQ ID NO: 2), respectively, were synthesized and cloned into the NdeI and BamHI sites of the pET28a-TEV expression vector. For initial testing, the construct containing the SiBP2 gene was used to transform E. coli BL21(DE3). After induction with IPTG, polypeptide expression recombinant SiBP2 was verified by SDS-PAGE. SEQ ID NO: 1 is represented by the following amino acid sequence: MGSSHHHHHHSSGENLYFQGHMAEPRLRTLEGHGGDHSISMSM HSSKAEKQAIEAAVEEDVAGPAKAAKLFKPKASKAGSMPDEASA KSAKMSMDTKSGKSEDAAAVDAKASKESHMSISGDMSMAKSHK AEAEDVTEMSMAKAGKDEASTEDMCMPFAKSDKEMSVKSKQGK TEMSVADAKASKESSMPSSKAAKIFKGKSGKSGSLSMLKSEKASS AHSLSMPKAEKVHSMSAGSGSGSIIII*

[0039] SEQ ID NO: 2 is represented by the following amino acid sequence: MGSSHHHHHHSSGENLYFQGHMASIADLAAANLSTEDSKSAQLISA DSSDDASDSSVESVDAASSDVSGSSVESVDVSGSSLESVDVSGSSLE SVDDSSEDSEEEELRILSSKKSGSYYSYGTKKSGSYSGYSTKKSASR RILSSKKSGSYSGYSTKKSGSRRILSSKKSGSYSGSKGSKRRILSSKK SGSYSGSKGSKRRNLSSKKSGSYSGSKGSKRRILSSKKSGSYSGSKG SKRRNLSSKKSGSYSGSKGSKRRILSGGLRGSM Example 2 - Purification of recombinant 6His-SiBP2 polypeptide and quartz microflotation tests

[0040] Purification of each recombinant enzyme was performed by an affinity chromatographic step on nickel resin (Ni-NTA Agarose, Qiagen). The chromatographic column was previously equilibrated with twice the column volume of 20 mM sodium phosphate buffer, pH 7.5, containing 300 mM NaCl and 5 mM imidazole. The polypeptide sample (cell lysis supernatant), previously filtered through a 0.45 µm membrane, was then injected. After sample application, the column was washed with 10 times the column volume of the same buffer used. The adsorbed polypeptides were eluted through an imidazole gradient in the buffer (500 mM imidazole). The fractions collected during elution were evaluated for purity by electrophoretic analyses by SDS-PAGE according to Laemmli (1970). Those fractions presenting the protein band with the expected size of the enzyme of interest were dialyzed in 1x PBS buffer with a 12-14 kDa retention membrane spectra / Por ® RC, according to the manufacturer's instructions at 4°C. The purified recombinant polypeptides were evaluated for their polypeptide content in a Nanodrop spectrophotometer (Thermo Scientific) using their molar extinction coefficient and subsequently stored at 4°C until use in the microflotation tests. Example 3 – Production and purification of the recombinant polypeptide 6His-SiBP2

[0041] For the production of the recombinant SiBP2 polypeptide in E. coli BL21(DE3), two constructs with the histidine tag (6His) at the amino-terminal portion of the SiBP2 polypeptide were produced, with one of the constructs lacking four isoleucines at the carboxy-terminus (6His-SiBP2-S4I). The 6His-SiBP2 and 6His-SiBP2-S4I polypeptides were purified by affinity chromatography, and the eluted fractions were analyzed by SDS-PAGE (Figure 1). As can be seen in this figure, this purification step removed most of the contaminating polypeptides, and the main components were the polypeptides of interest with a molecular mass of approximately 26 kDa and migrating between 25-35 kDa on the SDS-PAGE gel. These two pure polypeptides were subsequently used in microflotation assays to evaluate their potential to float quartz. Example 4 – Microflotation tests

[0042] In preliminary microflotation tests, it was verified that the two SiBP2 constructs in sodium phosphate buffer (PBS 1x) presented a buoyancy of approximately 70% for quartz (data not shown). In these tests, 400 µg (200 µL volume in PBS 1x buffer pH 7.4) of purified polypeptides were added and dialyzed in PBS buffer 1x pH 7.4. This resulted in a final concentration of approximately 3 mg / L of polypeptides in a volume of 140 mL of flotation medium.

[0043] Microflotation tests with the SiBP2 polypeptide revealed low foam production. However, the formation and stability of three-phase foams are also known to be related to other factors, such as equipment geometry, pulp rheology, hydrodynamics, and others. Therefore, the aforementioned behavior does not prevent its use as a bioreagent in quartz flotation. However, to improve quartz floatability using the SiBP2 polypeptide, tests were performed with the addition of different components to the final volume of 140 mL in the flotation medium, including ethanol 10 / 140,000 (v / v), ethyl dodecanoate 1 to 2 / 140,000 (v / v), and essential oils such as lemongrass or eucalyptus 1 to 2 / 140,000 (v / v) (Figure 1).

[0044] All these additives showed a positive effect on quartz floatability by SiBP2. The addition of lemongrass and eucalyptus essential oils showed a 90% higher floatability in all replicates. Statistical analysis of the results indicated no significant difference between SiBP2 with the different additives and the commercial bioreagent (Figure 1).

[0045] These preliminary results indicate that the use of the SiBP2 polypeptide in PBS 1x [400 µg in 200 / 140,000 (v / v)] optionally with additives such as essential oil 1 to 2 / 140,000 (v / v), is viable for quartz flotation because it does not use solvents in the SiBP2 polypeptide purification steps. Therefore, this polypeptide-based bioregent production route approaches a more renewable and sustainable system, in addition to being quite innovative, as there are no reports in the literature related to the use of polypeptides as a bioreagent for flotation.

Claims

MODIFIED CLAIMS Received by the International Bureau on 18 February 2025 (18.02.2025)

1. Composition, characterized by the fact that it comprises a polypeptide consisting of SEQ ID NO: 1 and a suitable vehicle, in which the vehicle comprises a PBS buffer 1x pH 7.

4.

2. Composition according to claim 1, characterized in that it may further comprise an additive and / or a starch depressant.

3. Composition according to claim 1 or 2, characterized in that the additive is an alcohol, ester or oil, preferably a vegetable additive.

4. Composition according to any one of claims 1 to 3, characterized in that the vegetable additive is ethanol 10 to 12 / 140,000 (v / v) (0.7% to 1.4%), ethyl dodecanoate 1 to 2 / 140,000 (v / v) (0.07% to 0.14%), essential oils such as lemongrass and / or eucalyptus essential oil 1 to 2 / 140,000 (v / v) (0.07% to 0.14%) in relation to the final volume of the flotation medium.

5. Composition according to any one of claims 1 to 4, characterized in that the vegetable additive is present in an amount in the range of about 1 / 140,000 (v / v) (0.07%) to about 10 / 140,000 (v / v) (0.7%) in the flotation medium.

6. Process for separating silica from a medium, characterized in that it comprises applying, to the medium in which the silica will be separated, a composition as defined in any one of claims 1 to 5.

7. Process, according to claim 6, characterized by the fact that the process of separating silica from a medium is beneficiation of an ore.

8. Process according to claim 6 or 7, characterized in that the ore to be processed is iron ore.

9. Process according to claim 7, characterized in that the beneficiation process is quartz flotation.

10. Process according to any one of claims 6 to 9, characterized in that a starch depressant can optionally be added to the reaction medium.

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