WO2020085219A1 - 新規ペプチド及びその利用方法 - Google Patents
新規ペプチド及びその利用方法 Download PDFInfo
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- WO2020085219A1 WO2020085219A1 PCT/JP2019/040997 JP2019040997W WO2020085219A1 WO 2020085219 A1 WO2020085219 A1 WO 2020085219A1 JP 2019040997 W JP2019040997 W JP 2019040997W WO 2020085219 A1 WO2020085219 A1 WO 2020085219A1
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- peptide
- ala
- arsenic
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- C22B3/00—Extraction of metal compounds from ores or concentrates by wet processes
- C22B3/02—Apparatus therefor
-
- C—CHEMISTRY; METALLURGY
- C22—METALLURGY; FERROUS OR NON-FERROUS ALLOYS; TREATMENT OF ALLOYS OR NON-FERROUS METALS
- C22B—PRODUCTION AND REFINING OF METALS; PRETREATMENT OF RAW MATERIALS
- C22B3/00—Extraction of metal compounds from ores or concentrates by wet processes
- C22B3/18—Extraction of metal compounds from ores or concentrates by wet processes with the aid of microorganisms or enzymes, e.g. bacteria or algae
-
- C—CHEMISTRY; METALLURGY
- C22—METALLURGY; FERROUS OR NON-FERROUS ALLOYS; TREATMENT OF ALLOYS OR NON-FERROUS METALS
- C22B—PRODUCTION AND REFINING OF METALS; PRETREATMENT OF RAW MATERIALS
- C22B3/00—Extraction of metal compounds from ores or concentrates by wet processes
- C22B3/20—Treatment or purification of solutions, e.g. obtained by leaching
- C22B3/22—Treatment or purification of solutions, e.g. obtained by leaching by physical processes, e.g. by filtration, by magnetic means, or by thermal decomposition
-
- C—CHEMISTRY; METALLURGY
- C22—METALLURGY; FERROUS OR NON-FERROUS ALLOYS; TREATMENT OF ALLOYS OR NON-FERROUS METALS
- C22B—PRODUCTION AND REFINING OF METALS; PRETREATMENT OF RAW MATERIALS
- C22B30/00—Obtaining antimony, arsenic or bismuth
- C22B30/04—Obtaining arsenic
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
- B03D—FLOTATION; DIFFERENTIAL SEDIMENTATION
- B03D2201/00—Specified effects produced by the flotation agents
- B03D2201/02—Collectors
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
- B03D—FLOTATION; DIFFERENTIAL SEDIMENTATION
- B03D2203/00—Specified materials treated by the flotation agents; Specified applications
- B03D2203/02—Ores
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P10/00—Technologies related to metal processing
- Y02P10/20—Recycling
Definitions
- the present invention relates to a novel peptide and a method of using the same.
- copper ores produced in copper mines are mainly sulfide ores. And, if this sulfide ore is roughly classified, it is mainly composed of minerals such as chalcocite (Cu 2 S) and copper indigo (CuS), and is mainly composed of secondary copper sulfide ore having a relatively high copper grade and chalcopyrite (CuFeS 2 ). There is a relatively low copper grade primary sulfide ore. In addition to these ores, minerals containing arsenic (for example, arsenopyrite) are also included.
- arsenic for example, arsenopyrite
- Arsenic is an element harmful to the environment, so if copper sulfide ore is used for smelting as it is with minerals containing arsenic, various problems will occur. Therefore, conventionally, various treatments for removing arsenic have been performed before smelting. For example, in Japanese Patent Laid-Open No. 2012-087400, measures such as roasting or the like to volatilize arsenic and remove arsenic from minerals in advance are taken.
- JP-A-2010-133304 proposes a method of using sodium thiosulfate as an inhibitor as a method of separating an arsenic mineral from a copper-containing material containing arsenic such as copper ore and copper concentrate.
- Japanese Patent Laid-Open No. 2011-156521 proposes a method of using a chelating agent such as polyethyleneamines as a suppressor at the time of flotation as a method for separating an arsenic mineral from a similar copper-containing material.
- WO2018 / 052134 discloses that phages that bind to arsenic spruce ore are obtained by repeatedly performing panning and screening.
- the patent document discloses a peptide that is expressed on the surface of a phage and that binds to arsenite.
- Patent Document 4 discloses that a specific peptide binds to arsenopyrite and the surface of the arsenopyrite is made hydrophilic by a bubble pickup test or the like.
- various factors that prevent binding with arsenopyrite are considered, and a peptide that can bind with arsenopyrite is required even under more severe conditions.
- the present invention includes the following inventions in one aspect.
- (Invention 1) A peptide having the following amino acid sequence. (TSNQ)-(LIVFA)-(ED)-(RKNMDCPQSETGWHY)-(LIVFA)-(RKNMMDCPQSETGWHY)-(LIVFA)-(LIVFA)-(RHK)-(TSNQ)-(TSNQ) ⁇ However, any one of at least one amino acid in () of the above formula is selected ⁇ (Invention 2) A peptide containing the following amino acid sequence: Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser (Invention 3) A peptide represented by the following amino acid sequence.
- Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser (Invention 4) A peptide comprising a sequence that is at least 75% identical to the following amino acid sequence. Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser (Invention 5) A peptide comprising a sequence that is at least 83% identical to the following amino acid sequence. Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser (Invention 6) A peptide comprising a sequence that is at least 90% identical to the following amino acid sequence.
- Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser (Invention 7) A peptide containing a sequence in which 1 to 5 amino acids are inserted, deleted, substituted and / or added in the following amino acid sequences.
- Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser (Invention 8) A composition for selecting and / or identifying an arsenic-containing mineral, the composition comprising the peptide according to any one of Inventions 1 to 7.
- invention 10 A nucleic acid having a sequence that is at least 90% identical to the sequence of the nucleic acid encoding the peptide according to any one of inventions 1 to 7.
- invention 11 A nucleic acid that hybridizes under stringent conditions with the complementary sequence of the nucleic acid encoding the peptide according to any one of inventions 1 to 7.
- invention 12 A microorganism having the peptide according to any one of inventions 1 to 7 displayed on its surface.
- Invention 13 A microorganism having the nucleic acid according to any one of Inventions 9 to 11.
- Invention 14 Fine particles having the peptide according to any one of inventions 1 to 7 on the surface.
- invention 15 A purification column comprising the peptide according to any one of inventions 1 to 7.
- invention 16 A collecting agent having the peptide according to any one of inventions 1 to 7.
- invention 17 An inhibitor comprising the peptide according to any one of inventions 1 to 7.
- invention 18 A method for extracting an arsenic-containing mineral, which uses the peptide according to any one of inventions 1 to 7.
- invention 19 A method for selecting and / or identifying arsenic-containing minerals using the peptide according to any one of inventions 1 to 7 or the composition according to invention 8.
- invention 20 A method according to invention 19, wherein A step of adding a microorganism having the peptide on the surface to a liquid in which arsenic-containing mineral particles are dispersed, Aggregating the arsenic-containing mineral particles and allowing them to settle; Collecting the aggregated and precipitated arsenic-containing mineral particles.
- invention 21 A method according to invention 19, wherein Immobilizing the peptide on a carrier, Introducing the carrier into a column, Passing a liquid in which arsenic-containing mineral particles are dispersed through the column.
- invention 22 A method according to invention 19, wherein Immobilizing the peptide on the microparticles, Adding the fine particles to a liquid in which arsenic-containing mineral particles are dispersed.
- invention 23 A method according to invention 19, wherein A method comprising performing flotation using the peptide.
- invention 24 A method according to invention 23, wherein The step of performing the flotation is Including a mixture of the arsenic-containing mineral, pyrite and / or chalcopyrite, and including the peptide or a microorganism containing the peptide as an inhibitor, This sorts the arsenic-containing minerals into tailings and pyrite and / or chalcopyrite into floating ores, The method.
- Invention 25 25.
- the method according to invention 24 or 25, wherein the step of performing flotation using the peptide comprises adding the peptide or a microorganism containing the peptide as an inhibitor, and then adding a collecting agent.
- the method comprising:
- the present invention utilizes peptides.
- the target mineral can be separated without requiring large-scale equipment as compared with the conventional method.
- the peptide used in the present invention enables efficient separation.
- the present invention can be applied to a method for separating a specific substance in one embodiment.
- Specific substances include minerals containing arsenic. More specifically, a copper mineral containing arsenic can be mentioned. Examples of copper minerals containing arsenic include arsenic spruce ore and arsenic tetrahedrite.
- the present invention can use peptides in one embodiment. More specifically, a peptide containing at least the following sequences can be used. Typically, a range defined by two numbers selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 (example: 1 or more and 10 or less, 5 to 20 amino acids) can be added to the N-terminal side and / or C-terminal side of the following amino acid sequence.
- the first sequence (2) is serine. It is a polar uncharged amino acid. Therefore, it is considered that similar effects can be obtained by substituting threonine, asparagine, and glutamine having similar properties. It is considered that similar substitution can be made for the 12th serine and the 11th threonine in the sequence (2).
- the second in sequence (2) is leucine.
- Leucine is a hydrophobic amino acid. Therefore, it is considered that the same effect can be obtained by substituting isoleucine, valine, phenylalanine, alanine, etc., which have similar properties. It is considered that similar substitution can be made for leucine at the 9th position in sequence (2) and alanine at the 5th, 7th, and 8th positions in sequence (2).
- the third in sequence (2) is aspartic acid. It is an acidic amino acid. Therefore, it is considered that similar effects can be obtained even if glutamic acid, which has similar properties, is substituted.
- the fourth in sequence (2) is glycine. Since the side chain of glycine is (-H), it is unlikely to be involved in a specific function by itself. Therefore, it is considered that the same effect can be obtained by substituting any natural amino acid for the glycine moiety. It is considered that similar substitution is possible for the glycine at the 6th position in sequence (2).
- the 10th in sequence (2) is arginine.
- Arginine has a basic side chain. Therefore, it is considered that the same effect can be obtained by substituting lysine and histidine, which have similar properties.
- the present invention includes, in one embodiment, a peptide containing the following amino acid sequence.
- a peptide containing the following amino acid sequence (2) Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser
- An arbitrary number of amino acids may be added to the N-terminal side and / or the C-terminal side of the amino acid sequence (2).
- a range defined by two numbers selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 can be added to the N-terminal side and / or the C-terminal side.
- this invention includes the peptide represented by the following 12 amino acid sequences in one embodiment.
- amino acid sequence (2) described above can exhibit the same function as the amino acid sequence (2) even if minor modifications (eg, amino acid insertion, substitution, addition) are made.
- a peptide which is 66% or more, 75% or more, 83% or more, 90% or more, 95% or more, 98% or more, or 99% or more identical to the amino acid sequence (2), or a peptide containing a sequence having said identity. Can also perform the same function.
- a method known in the art can be used. For example, it may be based on a numerical value determined by Blastp or the like used in a homology search of amino acids (or proteins) provided by BLAST (registered trademark).
- the present invention also includes, in one embodiment, a peptide containing a sequence in which 1 to 5 amino acids have been inserted, deleted, substituted and / or added in any one of the following amino acid sequences. Typically, it includes peptides containing sequences with 4 or less, 3 or less, or 2 or less amino acids inserted, deleted, substituted and / or added.
- the present invention also includes, in one embodiment, a composition containing the peptide described above. That is, the same function can be exhibited not only by using the above-mentioned peptide alone but also by a composition containing other components.
- the composition can contain any component as long as the function of the above-mentioned peptide is not impaired (buffer, sodium chloride, saccharides, etc.).
- the invention includes, in one embodiment, nucleic acids encoding the peptides described above.
- the nucleic acid may be DNA or RNA.
- the present invention may be, in one embodiment, a nucleic acid having a sequence complementary to the sense strand of the nucleic acid encoding the above-mentioned peptide.
- the present invention in one embodiment, has a sequence that is at least 80% or more, 85% or more, 90% or more, 95% or more, 98% or more identical to the nucleic acid sequence encoding the nucleic acid encoding the peptide described above. Includes nucleic acids.
- the method of calculating the sequence identity it can be calculated by using a known technique as in the case of the above-mentioned amino acid sequence. For example, it may be based on a numerical value determined when searching in BLAST Blastn or the like.
- the present invention includes, in one embodiment, a nucleic acid capable of hybridizing with a sequence complementary to the sense strand of the nucleic acid encoding the peptide described above. More specifically, it includes a nucleic acid that can hybridize under stringent conditions.
- stringent condition a standard known in the art can be used. For example, the criteria described in JP-A-2005-023831 may be used as the condition. Specifically, using a filter on which DNA is immobilized, hybridization is performed at 65 ° C. in the presence of 0.7 to 1.0 M sodium chloride, and then 0.1 to 2 times the concentration of SSC (saline- It means the conditions that can be identified by washing the filter under the condition of 65 ° C. using the sodium citrate solution (the composition of the SSC solution of 1 ⁇ concentration is 150 mM sodium chloride, 15 mM sodium citrate).
- nucleic acids are useful for producing a desired peptide through a genetic engineering technique.
- any of the above-described nucleic acids can be incorporated into an expression vector to express the target peptide in a large amount.
- the phage having the target peptide on the surface can be produced by using the phage display method described later.
- Microorganisms For example, by using a genetic engineering technique (for example, by introducing the above-mentioned nucleic acid into a gene of a microorganism), the microorganism can produce a large amount of a target peptide. Alternatively, a desired peptide can be expressed on the surface of a microorganism and the desired substance can be separated using the microorganism.
- the “microorganism” described in the present specification includes organisms belonging to the kingdoms of fungi, monella, or protozoa as described in the five kingdom theory. In addition, although it does not correspond to an organism in a strict sense, the “microorganism” described in the present specification also includes a virus. Typically, fungi, bacteria and viruses are used. Particularly preferred are those for which genetic engineering techniques have been established (eg, yeast, E. coli, lactic acid bacteria, bacteriophage, etc.). The present invention includes, in one embodiment, such a microorganism.
- microparticles encompasses microparticles having peptides on their surface.
- the peptide the above-mentioned peptides can be used.
- the fine particles include beads (eg, magnetic beads, glass beads, polymer beads, etc.) and carriers.
- the size of the fine particles is not particularly limited and may be appropriately adjusted depending on the application. Further, as a method for binding the peptide to the surface of the fine particles, a method known in the art can be used.
- the target substance can be separated by using the fine particles having the above-mentioned peptide on the surface.
- the target substance can be separated by binding it to a peptide and allowing it to precipitate using the method described below.
- Column chromatography can be mentioned as a method for separating a target substance.
- Column chromatography utilizes the fact that a column (functional group on the surface of a column) selectively binds to a specific substance.
- the above-mentioned peptide can be supported on a carrier. Then, this carrier can be introduced into the column. By using such a column, the target substance can be separated.
- Flotation agent for flotation Flotation is a method of separating fine particles by trapping them in bubbles.
- a scavenger can be used.
- the peptide can be bound to a known scavenger or foaming agent and used as a form that is easily trapped in air bubbles.
- the peptides of the invention hydrophilize the surface of certain minerals. As a result, it is possible to prevent a specific mineral from floating during flotation. Therefore, the peptide of the present invention can be used as an inhibitor. At this time, the peptide may be used alone, may be used in a form bound to a microorganism, or may be used in a form bound to a specific compound.
- the arsenic-containing minerals described above eg, arsenic spruce ore
- the peptide of the present invention can bind more specifically to an arsenic-containing mineral even in the presence of a substance that inhibits binding to the peptide (eg, impurities, surfactants, etc.).
- a microorganism can be used to separate substances (specifically, arsenic-containing minerals, more specifically, arsenopyrite).
- substances specifically, arsenic-containing minerals, more specifically, arsenopyrite.
- the microorganism any of the above-mentioned microorganisms can be used.
- a bacteriophage is mentioned.
- a nucleic acid sequence encoding the above-mentioned peptide can be introduced into a microorganism by a known genetic engineering technique and expressed on the surface of the microorganism. Then, the microorganism can be added to the liquid in which the mineral particles are dispersed.
- the amount of the microorganism added can be appropriately determined in consideration of various conditions such as the amount of mineral particles dispersed in the solution.
- the amount of mineral particles may be 10 10 pfu / mL to 10 18 pfu / mL with respect to 100 g / L. It is preferably 10 11 pfu / mL or more, more preferably 10 12 pfu / mL or more.
- the upper limit is preferably 10 16 pfu / mL or less, more preferably 10 15 pfu / mL or less.
- it may be 10 17 pfu / mL to 10 21 pfu / mL with respect to the amount of mineral particles of 3 g / L.
- it is 10 18 pfu / mL to 10 20 pfu / mL.
- the peptides on the surface of the microorganisms will bind to the mineral particles and agglomeration will occur. Then, it settles to the bottom of the solution. After that, the minerals that have settled to the bottom can be recovered.
- a column chromatography can be used to separate substances (specifically, arsenic-containing minerals, more specifically, arscopyrite).
- substances specifically, arsenic-containing minerals, more specifically, arscopyrite.
- the carrier can be introduced into a column for purification.
- the column is ready, the liquid in which the substance to be separated is dispersed is passed through the column. The substance then either binds into the column or is delayed in elution. Thereby, a specific substance can be separated.
- a substance (specifically, an arsenic-containing mineral, more specifically, arsenopyrite) can be separated using fine particles.
- the peptide described above can be immobilized on the surface of fine particles by a known method. Thereafter, the fine particles can be added to the liquid in which the mineral particles are dispersed. After adding the fine particles, if left for a while, the peptides on the surface of the fine particles are bound to the mineral particles and agglomeration occurs. Then, it settles to the bottom of the solution. After that, the minerals that have settled to the bottom can be recovered.
- magnetic beads can be used as the fine particles, and the magnetic particles can be used to recover the mineral particles without waiting for the sedimentation.
- the present invention separates substances (specifically, arsenic-containing minerals, more specifically, arsenopyrite) using a scavenger and / or a foaming agent. can do.
- a scavenger and / or a foaming agent is bound to the above-mentioned peptide by a known method.
- the bound scavenger is introduced into the solution and agitated (other chemicals are also introduced as appropriate) to generate bubbles.
- the mineral particles are introduced to trap the mineral particles in the bubbles. Thereby, the mineral particles can be collected.
- peptide in a form that allows it to function as a scavenger by binding to a chemical site that imparts hydrophobicity (eg, alkyl group, phenyl group, hydrophobic amino acid, etc.) and is easily trapped in bubbles.
- hydrophobicity eg, alkyl group, phenyl group, hydrophobic amino acid, etc.
- the peptides of this invention can be used as inhibitors.
- the inhibitor is a reagent that suppresses the floating property of a specific mineral in the flotation process.
- the peptide of the present invention may be used by being integrated with a microorganism. More specifically, the peptide of the present invention can be used in such a form that the peptide is displayed on the surface of a microorganism.
- the microorganism the microorganisms described in the section “4-1. Microorganism” can be used.
- the preferred microorganism is a phage and the more preferred microorganism is the M13 bacteriophage.
- the peptide of the present invention has a property of hydrophilizing the surface of an arsenic-containing mineral (eg, arsenopyrite), which causes the arsenic-containing mineral to form bubbles. It is thought to suppress trapping.
- an arsenic-containing mineral eg, arsenopyrite
- the present invention may be particularly useful in sorting with minerals that may be sorted into floating ores (eg, chalcopyrite, etc.).
- a suspension of an arsenic-containing mineral eg, arsenite
- a peptide and / or a microorganism containing the peptide can be charged with a peptide and / or a microorganism containing the peptide, and then a scavenger.
- the conditions of flotation are not particularly limited, but typically, the conditions can be carried out under the following conditions. Pulp concentration 50-600 (dry-g / L) Flotation time 5 to 30 minutes Flotation pH 3 or more and 10 or less Scavenger 5 to 100 g / t (weight of target mineral) Foaming agent 0.001-100g / t (floating solution)
- the amount of the peptide is not particularly limited, and the amount capable of exerting the effect of the inhibitor can be appropriately determined under the above-mentioned flotation conditions.
- an amount corresponding to the above peptide amount can be used.
- phage it is 10 10 pfu / L to 10 18 pfu / L (10 14 to 10 22 pfu / T).
- the scavenger acts to increase the hydrophobicity of the target mineral by selectively adsorbing it on the surface.
- the specific substance is not particularly limited, but is a commonly used / commercial collector for preferentially recovering copper sulfide minerals, specifically, xanthates, thionocarbamates, and mixtures thereof. More specifically, potassium amyl xanthate and the like can be mentioned.
- the amount of scavenger is 5-100 g / t. If it is less than 5 g / t, floating ore is difficult to obtain, which is not desirable, and if it exceeds 100 g / t, the effect will reach the ceiling, so there is no point in adding more.
- a foaming agent is a substance that dissolves in a solvent to stabilize the foam of the solution.
- Specific examples of the substance include, but are not limited to, Tween, methylisobutylcarbinol (MIBC), pine oil, and Aerofroth 70 (CYTEC).
- the amount of foaming agent is 0.001 to 100 g / t. If it is less than 0.001 g / t, it is not desirable because floating ore is difficult to obtain, and if it exceeds 100 g / t, the effect is reaching the ceiling, so it is meaningless to add more.
- the peptides described above have the selectivity that they bind to particular minerals particularly strongly and not to other minerals. More specifically, it binds strongly to arsenic-containing minerals (eg, arsenopyrite) and does not bind to other minerals (eg, chalcopyrite) (or the degree of bonding compared to arsenic-containing minerals). Is extremely low). Therefore, even with a mixture of a mineral containing arsenic and another mineral, the mineral containing arsenic can be separated and removed by using the method described above.
- the peptides described above can be used to identify minerals containing arsenic. For example, an arsenic-containing mineral can be detected by binding an identification marker (eg, a fluorescent molecule etc.) to the peptide of the present invention.
- an identification marker eg, a fluorescent molecule etc.
- the above-mentioned peptide can function as an inhibitor even in the presence of a scavenger and / or a foaming agent. That is, the above-mentioned peptide can hydrophilize the surface of a specific mineral. Alternatively, the above-mentioned peptides maintain the binding property with a specific mineral even in the presence of a surfactant. More preferably, the above-mentioned peptides maintain the selective binding property with a specific mineral even in the presence of a surfactant. These properties are particularly useful in the actual flotation environment because surfactants are often used in flotation. Examples of the surfactant include anionic, cationic, amphoteric, nonionic and the like. It is typically nonionic. Examples of the nonionic surfactant include Tween 20 and the like.
- Creating aforementioned peptide peptides can be prepared in a variety of ways.
- the DNA encoding the above-mentioned peptide can be incorporated into an expression vector, introduced into a microorganism or the like, and the peptide can be expressed in a large amount and recovered. Alternatively, it may be synthesized by an organic chemical method in addition to a genetic engineering method.
- the phage display method can be used when producing a phage (for example, M13 phage) displaying the above-mentioned peptide on the surface.
- a phage for example, M13 phage
- a microorganism displaying the desired peptide on its surface can be produced by a known genetic engineering technique.
- Example 1 Selection of arsenic spruce ore adsorbed phage by phage display method
- the phage display method was used for selection of peptide molecules adsorbed to arsenic spp. Specifically, an M13 bacteriophage library in which 12 amino acids were randomly bound was used, contacted with arsenic sulfite crushed to a particle size of 75 ⁇ m or less, and only the bacteriophage adsorbed on the arsenic sphalerite was recovered. Was infected with A. cerevisiae, proliferated again, and then contacted again with arsenic sulfite, and only the adsorbed phage was recovered. This adsorption / recovery operation (panning) was repeated several times, the DNA sequence of the selected phage was analyzed, and the amino acid sequence bound to the phage was identified.
- Panning was repeated 4 times with the pulp concentration of the arsenopyrite being brought into contact with 3000 ppm.
- a surfactant Teween (registered trademark) -20
- concentration during panning for the concentration, 1st time 0.1%, 2nd time 0.5%, 3rd time 0.7%, 4th time 1.0%.
- Example 2 ELISA analysis of arsenopyrite binding phage Phosphosporite and A56-page selected in Example 1 were bound to arsenopyrite by an ELISA method (Enzyme-Linked ImmunoSorbent Assay).
- ELISA method Enzyme-Linked ImmunoSorbent Assay
- ABTS 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
- Example 3 Bubble Pick Up test using peptides (arsenopyrite)
- the A56-page of Examples 1-2 was used in the Bubble Pick Up test. More specifically, first, the arsenic spruce ore was suspended in pure water so that the concentration was 100 g / L, and the pH value was adjusted to 7. Furthermore, MIBC was also added as a foaming agent (final concentration 10 ⁇ L / L). Next, a sample in which A56-phase (concentration after addition of 10 10 to 10 18 pfu / L) was added to the suspension and a sample in which this was not added were prepared. Then, these were stirred with a vortex mixer for 1 minute and left to stand for 5 minutes. Next, after the addition of A56-page, the scavenger Xanthate was added. In another example, the scavenger Xanthate was added prior to the addition of A56-page.
- Adhesion probability (%) (number of times minerals have adhered / 30) x 100%
- Example 4 Comparative test of peptide binding ability in the presence of a surfactant The following two types of peptides were prepared. Fluorescein was bound to these peptides at the N-terminal side as a fluorescent label. (1) Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser (Peptide obtained from A56-page, hereinafter referred to as A56 peptide) (2) Asn-Pro-Glu-His-Ala-Ala-Phe-Ser-Pro-Val-Thr-Val (a peptide obtained from A710-page (see WO2018 / 052134), hereinafter referred to as A710 peptide)
- Arsenopyrite was fixed to the bottom of a 96-well plate (mineral concentration 3 g / L). A peptide-containing buffer solution was introduced into each well to bond the peptide with arsenic spruce ore. Then, the supernatant was sucked up and washed 3 times with a peptide-free buffer solution. After washing, fluorescence measurement was performed. A citrate buffer solution (concentration 50 mM) adjusted to pH 3 was used as the buffer solution. The concentration of peptide in the buffer was adjusted to 100 ng / mL.
- Two types of peptide-containing buffer solutions were prepared. Specifically, a buffer solution containing Tween 20 and a buffer solution not containing Tween 20 were prepared.
- the amount of Tween 20 in the A56 peptide was large and the conditions were even more severe. Nonetheless, the A710 peptide significantly reduced the binding ability, while the A56 peptide maintained the binding ability. These data indicate that the A56 peptide may be useful in flotation using foaming agents and the like.
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Abstract
Description
また、特開2011-156521号では、同様の含銅物からヒ素鉱物を分離する方法として、浮遊選鉱時にポリエチレンアミン類等のキレート剤を抑制剤として使用する方法を提案している。
更に、国際公開第2018/052134号では、パニングを繰り返してスクリーニングすることで、硫砒銅鉱に結合するファージを得ることを開示している。更に該特許文献では、ファージの表面に発現し、且つ硫砒銅鉱に結合するペプチドを開示している。
以下のアミノ酸配列を有するペプチド。
(TSNQ)-(LIVFA)-(ED)-(RKNMDCPQSETGWHY)-(LIVFA)-(RKNMDCPQSETGWHY)-(LIVFA)-(LIVFA)-(LIVFA)-(RHK)-(TSNQ)-(TSNQ)
{ただし、上記式の()中の少なくとも1種のアミノ酸のいずれか1つが選択される}
(発明2)
以下のアミノ酸配列を含むペプチド。
Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser
(発明3)
以下のアミノ酸配列で表されるペプチド。
Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser
(発明4)
以下のアミノ酸配列と少なくとも75%同一である配列を含むペプチド。
Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser
(発明5)
以下のアミノ酸配列と少なくとも83%同一である配列を含むペプチド。
Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser
(発明6)
以下のアミノ酸配列と少なくとも90%同一である配列を含むペプチド。
Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser
(発明7)
以下のアミノ酸配列において、1~5個のアミノ酸が挿入、欠失、置換及び/又は付加された配列を含むペプチド。
Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser
(発明8)
ヒ素含有鉱物を選別及び/又は識別するための組成物であって、発明1~7のいずれか1つに記載のペプチドを含む、該組成物。
(発明9)
発明1~7のいずれか1つに記載のペプチドをコードする核酸。
(発明10)
発明1~7のいずれか1つに記載のペプチドをコードする核酸の配列と少なくとも90%以上同一の配列を有する核酸。
(発明11)
発明1~7のいずれか1つに記載のペプチドをコードする核酸の相補配列とストリンジェントな条件下でハイブリダイズする核酸。
(発明12)
発明1~7のいずれか1つに記載のペプチドを表面に提示した微生物。
(発明13)
発明9~11のいずれか1つに記載の核酸を有する微生物。
(発明14)
発明1~7のいずれか1つに記載のペプチドを表面に有する微粒子。
(発明15)
発明1~7のいずれか1つに記載のペプチドを有する精製カラム。
(発明16)
発明1~7のいずれか1つに記載のペプチドを有する捕収剤。
(発明17)
発明1~7のいずれか1つに記載のペプチドを有する抑制剤。
(発明18)
発明1~7のいずれか1つに記載のペプチドを使用する、ヒ素含有鉱物を抽出するための方法。
(発明19)
発明1~7のいずれか1つに記載のペプチド又は発明8の組成物を使用する、ヒ素含有鉱物を選別及び/又は識別するための方法。
(発明20)
発明19に記載の方法であって、
前記ペプチドを表面に有する微生物を、ヒ素含有鉱物粒子が分散した液に添加するステップと、
前記ヒ素含有鉱物粒子を凝集させ、沈降させるステップと、
前記凝集及び沈降したヒ素含有鉱物粒子を回収するステップと
を含む方法。
(発明21)
発明19に記載の方法であって、
前記ペプチドを担体に固定するステップと、
前記担体をカラムに導入するステップと、
ヒ素含有鉱物粒子が分散した液を前記カラムに通過させるステップと
を含む方法。
(発明22)
発明19に記載の方法であって、
前記ペプチドを微粒子に固定するステップと、
ヒ素含有鉱物粒子が分散した液に、前記微粒子を添加するステップと
を含む方法。
(発明23)
発明19に記載の方法であって、
前記ペプチドを用いた浮遊選鉱を行うステップ
を含む方法。
(発明24)
発明23に記載の方法であって、
前記浮遊選鉱を行うステップは、
前記ヒ素含有鉱物と、黄鉄鉱及び/又は黄銅鉱との混合物を投入すること、及び
前記ペプチド又は前記ペプチドを含む微生物を抑制剤として投入すること
を含み、
これにより前記ヒ素含有鉱物を尾鉱へ選別し、黄鉄鉱及び/又は黄銅鉱を浮鉱へと選別する、
該方法。
(発明25)
発明24に記載の方法であって、前記ヒ素含有鉱物が硫砒銅鉱であり、前記微生物がファージである、該方法。
(発明26)
発明24又は25に記載の方法であって、前記ペプチドを用いた浮遊選鉱を行うステップが、前記ペプチド又は前記ペプチドを含む微生物を抑制剤として投入した後で、更に、捕収剤を投入することを含む、該方法。
本発明は、一実施形態において、特定の物質を分離する方法に適用することができる。特定の物質として、ヒ素を含む鉱物が挙げられる。更に具体的には、ヒ素を含む銅鉱物が挙げられる。ヒ素を含む銅鉱物としては、硫砒銅鉱や砒四面銅鉱等が挙げられる。
上述した物質を分離するため、本発明は、一実施形態において、ペプチドを用いることができる。より具体的には、少なくとも以下の配列を含むペプチドを用いることができる。典型的には、1、2、3、4、5、6、7、8、9、10、15、20のうちから選択される2つの数で規定される範囲(例:1以上10以下、5以上20以下)のアミノ酸を下記のアミノ酸配列のN末端側及び/又はC末端側に付加することができる。
{ただし、上記式の()中の少なくとも1種のアミノ酸のいずれか1つが選択される}
(2)Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser
(2)Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser
アミノ酸配列(2)のN末端側及び/又はC末端側には、任意の数のアミノ酸が付加されてもよい。典型的には、1、2、3、4、5、6、7、8、9、10、15、20のうちから選択される2つの数で規定される範囲(例:1以上10以下、5以上20以下)のアミノ酸をN末端側及び/又はC末端側に付加することができる。
(2)Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser
(2)Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser
本発明は、一実施形態において、上述したペプチドをコードする核酸を包含する。核酸は、DNAでもRNAでもよい。また、本発明は、一実施形態において、上述したペプチドをコードする核酸のセンス鎖に対して相補的な配列を有する核酸であってもよい。
上述したペプチド及び/又は核酸は、様々な形で応用することができる。
例えば、遺伝子工学的な手法を用いて(例えば、微生物の遺伝子に上述した核酸を導入して)、微生物に目的のペプチドを大量に生成させることができる。あるいは、微生物の表面に目的のペプチドを発現させて、該微生物を利用して、目的の物質を分離することができる。本明細書で述べる「微生物」には、五界説で述べるところの菌界、モネラ界、又は原生生物界に属する生物が含まれる。また、厳密な意味では生物には該当しないものの、本明細書で述べる「微生物」には、ウイルスも含まれる。典型的には、真菌、細菌、ウイルスを用いる。特に好ましいのは、遺伝子工学的な手法が確立された物である(例:酵母、E.coli、乳酸菌、バクテリオファージなど)。本発明は、一実施形態において、このような微生物を包含する。
本発明は、一実施形態において、ペプチドを表面に有する微粒子を包含する。ペプチドは、上述したペプチドを用いることができる。また、微粒子は、ビーズ(例:磁気ビーズ、ガラスビーズ、高分子ビーズなど)、担体等が挙げられる。微粒子の大きさについては、特に限定されず、用途に応じて適宜調整すればよい。また、微粒子の表面にペプチドを結合させる手法については、当分野で公知の手法を用いることができる。
目的の物質を分離する方法としてカラムクロマトグラフィーが挙げられる。カラムクロマトグラフィーは、カラム(カラム表面の官能基)が特定の物質に選択的に結合することを利用する。本発明の一実施形態では、上述したペプチドを担体に担持させることができる。そして、この担体をカラムに導入することができる。こうしたカラムを使用することにより、目的の物質を分離することができる。
浮遊選鉱(浮選)は、微粒子を気泡にトラップさせることにより分離する方法である。この際に、捕収剤を使用することができる。本発明の一実施形態では、ペプチドを公知の捕収剤や起泡剤に結合し、気泡にトラップしやすい形態として用いることができる。あるいは、当該ペプチドを、疎水性を付与する化学的部位(例、アルキル基、フェニル基、疎水性のアミノ酸等)と結合することで捕収剤として機能させ、気泡にトラップさせやすい形態として用いることができる。これにより、目的の物質を気泡にトラップすることができ、結果として分離することができる。
別の一実施形態では、本発明のペプチドは、特定の鉱物の表面を親水化させる。これにより、浮遊選鉱の際に、特定の鉱物が浮遊するのを抑制することができる。従って、本発明のペプチドは、抑制剤として使用することができる。この際に、ペプチド単独で用いてもよいし、微生物に結合した態様で使用してもよいし、特定の化合物と結合した形で使用してもよい。
上述した応用形態に関する方法を以下に具体的に説明する。
上述した応用形態はいずれも所定の物質を分離することに関する。例えば、上述したヒ素含有鉱物(例えば、硫砒銅鉱)を分離することができる。一実施形態において、ペプチドとの結合を阻害する物質(例えば、不純物、界面活性剤など)の存在下でも、本発明のペプチドは、ヒ素含有鉱物とより特異的に結合することができる。
本発明は、一実施形態において、微生物を用いて、物質(具体的には、ヒ素含有鉱物、より具体的には硫砒銅鉱)を分離することができる。微生物としては、上述した微生物であれば、いずれも用いることができる。典型的にはバクテリオファージが挙げられる。
本発明は、一実施形態において、カラムクロマトグラフィーを用いて、物質(具体的には、ヒ素含有鉱物、より具体的には硫砒銅鉱)を分離することができる。方法としては、まず、上述したペプチドを、公知の手法により、担体に固定させることができる。その後、その担体を精製用のカラムに導入することができる。前記カラムが準備できたら、分離対象の物質が分散した液を、前記カラムの中に通す。すると、前記物質は、カラムの中に結合するか、又は溶出が遅れる。これにより、特定の物質を分離することができる。
本発明は、一実施形態において、微粒子を用いて、物質(具体的には、ヒ素含有鉱物、より具体的には硫砒銅鉱)を分離することができる。まず、上述したペプチドを、公知の手法により、微粒子表面に固定させることができる。その後、鉱物粒子が分散した液に、微粒子を添加することができる。微粒子を添加した後、暫く放置すると、微粒子表面にあるペプチドが鉱物粒子と結合し、凝集が起こる。そして、溶液の底に沈降する。その後、底に沈降した鉱物を回収することができる。あるいは、微粒子として磁気ビーズを用いることができ、沈降することを待つことなく、磁力を用いて、鉱物粒子を回収することができる。
本発明は、一実施形態において、捕収剤及び/又は起泡剤を用いて、物質(具体的には、ヒ素含有鉱物、より具体的には硫砒銅鉱)を分離することができる。具体的には、捕収剤及び/又は起泡剤を、公知の方法により、上述したペプチドと結合させる。そして、結合させた捕収剤を溶液に導入して撹拌させ(適宜他の薬剤も導入し)、気泡を発生させる。その後、鉱物粒子を導入し、該鉱物粒子を気泡にトラップさせる。これにより、鉱物粒子を回収することができる。あるいは、当該ペプチドを、疎水性を付与する化学的部位(例、アルキル基、フェニル基、疎水性のアミノ酸等)と結合することで捕収剤として機能させ、気泡にトラップさせやすい形態として用いることができる。
また、本発明のペプチドは微生物と一体化させて使用してもよい。より具体的には、本発明のペプチドは、微生物の表面にペプチドを提示させるような形態で使用することができる。微生物については、「4-1.微生物」の項で述べた微生物を使用することができる。好ましい微生物はファージであり、より好ましい微生物はM13バクテリオファージである。
パルプ濃度 50~600(dry-g/L)
浮選時間 5~30分
浮選pH 3以上10以下
捕収剤 5~100g/t(対象鉱物重量)
起泡剤 0.001~100g/t(浮遊選鉱溶液)
上述したペプチドは、特定の鉱物に特に強く結合し、他の鉱物には結合しないという選択性を有する。より具体的には、ヒ素を含む鉱物(例:硫砒銅鉱)には強く結合し、他の鉱物(例えば、黄銅鉱)には、結合しない(又は、ヒ素を含む鉱物の場合と比べて結合度合いが著しく低い)という性質を有する。従って、ヒ素を含む鉱物と他の鉱物との混合物であっても、上述した方法を用いることにより、ヒ素を含む鉱物を分離・除去することができる。あるいは、上述したペプチドは、ヒ素を含む鉱物を識別するために用いることができる。例えば、本発明のペプチドに識別マーカー(例:蛍光分子など)を結合させることにより、ヒ素を含む鉱物を検出することができる。
上述したペプチドは、様々な方法で製造することができる。上述したペプチドをコードするDNAを、発現ベクターに組み込んで、微生物等に導入し、大量にペプチドを発現させて回収することができる。あるいは、遺伝子工学的な手法のほか、有機化学的な方法により合成してもよい。
硫砒銅鉱に吸着するペプチド分子の選択としては、ファージディスプレイ法を用いた。具体的には、アミノ酸12個がランダムに結合したM13バクテリオファージライブラリーを用い、粒度75μm以下に粉砕した硫砒銅鉱と接触させ、硫砒銅鉱に吸着したバクテリオファージのみを回収し、回収したファージについて大腸菌に感染させ再度増殖後、再び硫砒銅鉱に接触させ、吸着したファージのみを回収した。この吸着・回収操作(パニング)を数回繰り返し、選択されたファージのDNA配列を解析し、ファージに結合したアミノ酸配列を特定した。
Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser
以後、上記に示すペプチドが結合したファージをA56-phageと呼ぶ。
硫砒銅鉱と実施例1にて選択したA56-phageについて、ELISA法(Enzyme-Linked ImmunoSorbent Assay、酵素結合免疫吸着法)により、硫砒銅鉱との結合量を測定した。具体的には硫砒銅鉱3000mg/Lを懸濁した液を96穴マイクロプレートの各ウェルに添加し、pH7の条件下でそれぞれのウェルに各ファージを添加後、未結合のファージを洗浄した。さらにこの懸濁液に酵素(ペルオキシダーゼ)標識した抗M13ファージ抗体を添加したのち、未結合の抗ファージ抗体を洗浄した。ここに酵素の基質となる2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)diammonium salt(ABTS)を添加し、青色の発色をマイクロプレートリーダーで波長405nmにて吸光度測定した。更に、これらの手順を、黄銅鉱に変更したうえで行った。
従って、A56-phageが、黄銅鉱よりも硫砒銅鉱に、より特異的に結合することが示された。このことにより、黄銅鉱と硫砒銅鉱との存在下で、A56-phageによる硫砒銅鉱の選別に有用であることが示された。
実施例1~2のA56-phageをBubble Pick Up試験に用いた。より具体的には、最初に、硫砒銅鉱の濃度が100g/Lになるように純水に懸濁し、pHの値を7に調整した。更には、起泡剤としてMIBCも添加した(最終濃度10μL/L)。次に、懸濁液にA56-phage(添加後の濃度1010~1018pfu/L)を投入したサンプル、及びこれを投入しないサンプルを用意した。そして、これらを1分間ボルテックスミキサーで撹拌し、5分間静置した。次に、A56-phageの添加の後に、捕収剤Xanthateを添加した。別の実施例では、A56-phageの添加の前に、捕収剤Xanthateを添加した。
付着確率(%)=(鉱物が付着した回数÷30)×100%
以下の二種類のペプチドを準備した。これらのペプチドには、蛍光標識として、FluoresceinをN末端側に結合させた。
(1)Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser(A56-phageから得られたペプチド、以下A56ペプチドと称する)
(2)Asn-Pro-Glu-His-Ala-Ala-Phe-Ser-Pro-Val-Thr-Val(A710-phageから得られたペプチド(WO2018/052134号参照)、以下A710ペプチドと称する)
Claims (26)
- 以下のアミノ酸配列を有するペプチド。
(TSNQ)-(LIVFA)-(ED)-(RKNMDCPQSETGWHY)-(LIVFA)-(RKNMDCPQSETGWHY)-(LIVFA)-(LIVFA)-(LIVFA)-(RHK)-(TSNQ)-(TSNQ)
{ただし、上記式の()中の少なくとも1種のアミノ酸のいずれか1つが選択される} - 以下のアミノ酸配列を含むペプチド。
Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser - 以下のアミノ酸配列で表されるペプチド。
Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser - 以下のアミノ酸配列と少なくとも75%同一である配列を含むペプチド。
Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser - 以下のアミノ酸配列と少なくとも83%同一である配列を含むペプチド。
Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser - 以下のアミノ酸配列と少なくとも90%同一である配列を含むペプチド。
Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser - 以下のアミノ酸配列において、1~5個のアミノ酸が挿入、欠失、置換及び/又は付加された配列を含むペプチド。
Ser-Leu-Asp-Gly-Ala-Gly-Ala-Ala-Leu-Arg-Thr-Ser - ヒ素含有鉱物を選別及び/又は識別するための組成物であって、請求項1~7のいずれか1項に記載のペプチドを含む、該組成物。
- 請求項1~7のいずれか1項に記載のペプチドをコードする核酸。
- 請求項1~7のいずれか1項に記載のペプチドをコードする核酸の配列と少なくとも90%以上同一の配列を有する核酸。
- 請求項1~7のいずれか1項に記載のペプチドをコードする核酸の相補配列とストリンジェントな条件下でハイブリダイズする核酸。
- 請求項1~7のいずれか1項に記載のペプチドを表面に提示した微生物。
- 請求項9~11のいずれか1項に記載の核酸を有する微生物。
- 請求項1~7のいずれか1項に記載のペプチドを表面に有する微粒子。
- 請求項1~7のいずれか1項に記載のペプチドを有する精製カラム。
- 請求項1~7のいずれか1項に記載のペプチドを有する捕収剤。
- 請求項1~7のいずれか1項に記載のペプチドを有する抑制剤。
- 請求項1~7のいずれか1項に記載のペプチドを使用する、ヒ素含有鉱物を抽出するための方法。
- 請求項1~7のいずれか1項に記載のペプチド又は請求項8の組成物を使用する、ヒ素含有鉱物を選別及び/又は識別するための方法。
- 請求項19に記載の方法であって、
前記ペプチドを表面に有する微生物を、ヒ素含有鉱物粒子が分散した液に添加するステップと、
前記ヒ素含有鉱物粒子を凝集させ、沈降させるステップと、
前記凝集及び沈降したヒ素含有鉱物粒子を回収するステップと
を含む方法。 - 請求項19に記載の方法であって、
前記ペプチドを担体に固定するステップと、
前記担体をカラムに導入するステップと、
ヒ素含有鉱物粒子が分散した液を前記カラムに通過させるステップと
を含む方法。 - 請求項19に記載の方法であって、
前記ペプチドを微粒子に固定するステップと、
ヒ素含有鉱物粒子が分散した液に、前記微粒子を添加するステップと
を含む方法。 - 請求項19に記載の方法であって、
前記ペプチドを用いた浮遊選鉱を行うステップ
を含む方法。 - 請求項23に記載の方法であって、
前記浮遊選鉱を行うステップは、
前記ヒ素含有鉱物と、黄鉄鉱及び/又は黄銅鉱との混合物を投入すること、及び
前記ペプチド又は前記ペプチドを含む微生物を抑制剤として投入すること
を含み、
これにより前記ヒ素含有鉱物を尾鉱へ選別し、黄鉄鉱及び/又は黄銅鉱を浮鉱へと選別する、
該方法。 - 請求項24に記載の方法であって、前記ヒ素含有鉱物が硫砒銅鉱であり、前記微生物がファージである、該方法。
- 請求項24又は25に記載の方法であって、前記ペプチドを用いた浮遊選鉱を行うステップが、前記ペプチド又は前記ペプチドを含む微生物を抑制剤として投入した後で、更に、捕収剤を投入することを含む、該方法。
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