JP2010131587A - Asbestos adsorbing material and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an asbestos adsorbing material capable of improving removal effect of asbestos and preventive effect of health hazard, and also to provide its use. <P>SOLUTION: The asbestos adsorbing material includes a carrier and asbestos-binding protein carried on the carrier. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an asbestos adsorbent using an asbestos-binding protein and use thereof.

  Asbestos (asbestos) is a stable substance excellent in heat resistance, wear resistance and electrical insulation, and has been widely used as, for example, a building material. However, in recent years, it has become clear that continuing to inhale asbestos into the body causes a health hazard called mesothelioma or asbestosis. For this reason, in buildings and the like in which asbestos is used, measures for preventing asbestos from being scattered and measures for removing asbestos are currently being performed. In addition, many buildings using asbestos have been built so far, and it is considered that it takes a long time to complete the above treatment.

  Asbestos is a fibrous hydrous silicate mineral having flexibility, and is easily scattered in the atmosphere. Therefore, in a building where asbestos is used, there is a risk of inhaling asbestos scattered in the atmosphere. Moreover, when it is going to remove asbestos from these buildings, the danger that asbestos will disperse | distribute in air | atmosphere further increases in the work process. Therefore, it can be said that workers handling asbestos have a very high risk of sucking asbestos. Therefore, various methods have been conventionally used as methods for preventing health damage caused by asbestos.

  For example, traditionally, workers who handle asbestos have been working in a strictly shielded room while wearing a protective garment that meets certain standards, as well as peeling off asbestos deposited on the floor. By sucking in with a suction device, health damage caused by asbestos is prevented. At this time, a method for preventing the asbestos from being scattered by using a surfactant and an organic paste as well as adding moisture to the object to be treated (for example, a building material containing asbestos) ( For example, see Patent Document 1).

  In addition, there has also been a method for preventing asbestos from being scattered outside the work environment, that is, into the atmosphere by actively collecting and removing asbestos scattered in the work environment with a dust collection / exhaust device having a HEPA filter. (For example, refer nonpatent literature 1).

  In recent years, proteins having the ability to bind to asbestos have been discovered, but at present, these proteins have not actually been used for removing asbestos (see, for example, Patent Document 2).

Japanese Laid-Open Patent Publication No. 06-049391 (Release Date: February 22, 1994) WO2007 / 055243A1 (International publication date: May 18, 2007)

"Exposure prevention manual for asbestos dust" in the construction and other work of building demolition, Construction and Industrial Accident Prevention Association Planning and Development Division, August 9, 2005, first edition issued

  However, the conventional method has a problem that the effect of removing asbestos and the effect of preventing health damage are low.

  For example, conventional protective clothing (eg, masks), suction devices (filters within the device), and filters as described above adsorb asbestos by non-specific physical adsorption forces, thereby They are protecting the human body and removing asbestos.

  However, the above-mentioned conventional method using non-specific physical adsorption force has improved the asbestos removal effect by reducing the pores of the filter. There is a problem that the holes are easily clogged.

  Further, as described above, since the physical adsorption force is weak, even if the asbestos can be captured by the filter, the captured asbestos is easily scattered from the filter by vibrations or the like. Therefore, the above-described conventional methods also have problems that the effect of preventing health damage is lowered and the effect of removing asbestos is lowered.

  In addition, since the technique of the said patent document 2 is not a technique for removing asbestos, the removal effect of asbestos and the prevention effect of a health hazard cannot be expected.

  The present invention has been made in view of the above-mentioned conventional problems, and an object thereof is to provide an asbestos adsorbent capable of enhancing the effect of removing asbestos and preventing health damage, and use thereof. It is in.

  As a result of intensive studies in view of the above problems, the present inventors have found that among proteins produced by microorganisms and the like, there are proteins that specifically bind to asbestos (for example, Akio Kuroda et al. , Biotechnology and Bioengineering, 2008, 99 (2), 285-289). As a result of further studies, the present invention was completed by finding that an asbestos adsorbent capable of effectively adsorbing asbestos can be produced by providing the protein on a carrier.

  That is, the asbestos adsorbent of the present invention is characterized by comprising a carrier and an asbestos-binding protein carried on the carrier in order to solve the above-mentioned problems.

  In the asbestos adsorbent of the present invention, the asbestos-binding protein comprises OmpC, OmpA, DksA, HlpA, YgiW, H-NS, CspA-2, Cgl0974, TTC0984, GatZ, L1, L5, S1, S4 and S7. It is preferably at least one protein selected from the group.

In the asbestos adsorbent of the present invention, the asbestos-binding protein is preferably a protein described in any of (I) to (III) below. That means
(I) a protein comprising the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29;
(II) One or several amino acid residues in the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29 Is a protein having an asbestos-binding property consisting of an amino acid sequence substituted, deleted, inserted and / or added;
(III) Amino acids having 55% or more homology with the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29 A protein comprising a sequence and having asbestos binding properties.

  In the asbestos adsorbent of the present invention, the carrier preferably has a cotton shape, a filter paper shape, a sheet shape, a honeycomb shape, a granular shape, a powder shape, a mesh shape, a porous shape, or a gel shape.

  In the asbestos adsorbent of the present invention, a layer for adjusting the humidity of the surface of the carrier is preferably provided on the surface of the carrier.

  In the asbestos adsorbent of the present invention, the layer is preferably formed by a water-absorbing and hygroscopic polymer provided on the surface of the carrier, or a first functional group introduced on the surface of the carrier. .

  In the asbestos adsorbent of the present invention, the polymer is a polyacrylic acid polymer, a polyacrylate polymer, a polyamide polymer, a polyalcohol polymer, a polyether polymer, a polyvinylpyrrolidone polymer, a polycarboxylic acid polymer, It is preferably at least one selected from the group consisting of a polyethyleneimine polymer, a polyvinyl pyridine polymer, a maleic acid polymer, a polyvinyl phosphonic acid, a polyvinyl sulfonic acid, a polysaccharide, and a polyamino acid.

  In the asbestos adsorbent of the present invention, the first functional group is preferably at least one selected from the group consisting of a carboxyl group, a derivative of a carboxyl group, an amide group, an amino group, and a hydroxyl group.

  In the asbestos adsorbent of the present invention, the asbestos-binding protein is preferably immobilized on the carrier via a second functional group, a metal ion, or a linker.

  In the asbestos adsorbent of the present invention, the second functional group is selected from the group consisting of an aldehyde group, an amino group, a carboxyl group, a hydroxyl group, a hydrazide group, a carbodiimide group, an epoxy group, a thiol group, an anhydrous ring, and a maleimide group. It is preferably at least one functional group selected.

  In the asbestos adsorbent of the present invention, the linker is maleimide, N-hydroxysuccinimidyl ester, imide ester, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N- (p-maleimidophenyl) isocyanate, It is preferably at least one compound selected from the group consisting of nitrilotriacetic acid, glutathione, and maltose.

  The asbestos adsorbent of the present invention is preferably for adsorbing asbestos in the gas phase.

  The asbestos removal method of the present invention is characterized by using the asbestos adsorbent in order to solve the above-mentioned problems.

  As described above, the asbestos adsorbent of the present invention comprises a carrier and an asbestos-binding protein carried on the carrier.

  Moreover, the asbestos removal method of the present invention is a method using the asbestos adsorbent of the present invention.

  Therefore, it is possible to efficiently adsorb asbestos and to prevent the once adsorbed asbestos from scattering again.

  Further, according to the present invention, there is an effect that asbestos can be sufficiently captured even if the pores of the filter are designed to be large.

  Moreover, if it is this invention, there exists an effect that generation | occurrence | production of the health hazard of the worker who handles asbestos can be prevented more reliably.

It is an electrophoretic diagram which shows DksA protein refine | purified from DksA protein contained in a DksA cell disruption liquid, and the said DksA cell disruption liquid.

  An embodiment of the present invention will be described below, but the present invention is not limited to this.

  The asbestos adsorbent of the present embodiment includes a carrier, and an asbestos-binding protein is supported on the carrier.

  The specific configuration of the carrier is not particularly limited. For example, the carrier is preferably insoluble in water, various organic solvents, and fats and oils. That is, the carrier is preferably physically and chemically stable. The carrier is preferably one that does not affect the binding of asbestos to the asbestos-binding protein.

  The shape of the carrier is not particularly limited, and can be a desired shape as appropriate. For example, the shape may be cotton (for example, non-woven fabric), filter paper, sheet, honeycomb, granular, powder, mesh (for example, woven fabric), porous, or gel. Is preferred. More specifically, the carrier is preferably, for example, a woven fabric, a nonwoven fabric, a HEPA filter, a filter paper, a depth filter, a membrane filter, or a column carrier. According to the above configuration, a large amount of asbestos-binding protein can be supported on the surface of the carrier, so that asbestos can be adsorbed more effectively.

  The material for the woven fabric, nonwoven fabric, depth filter and the like is not particularly limited. For example, polypropylene, polystyrene, rayon, polyester, polyethylene, polyolefin, modacrylic, nylon, cupra, vinylon, acrylic, aramid, glass, cellulose, Cotton, wool, silk, hemp or pulp is preferred. Further, it is more preferable to use a carrier composed of a plurality of these materials.

  Further, the material of the filter paper is not particularly limited, but for example, cellulose, glass, polytetrafluoroethylene (PTFE), or silica is preferable.

  The material for the membrane filter is not particularly limited. For example, nylon, Teflon (registered trademark) (for example, PTFE, polyvinylidene fluoride (PVDF), etc.), polycarbonate, polyethersulfone (PES), cellulose mixture An ester or cellulose acetate is preferred.

  In addition, the column carrier and other carrier materials are not particularly limited. For example, celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics and other inorganic substances, cellulose powder, ceramics Preferred are organic polymers such as powder, polyvinyl alcohol (particularly cross-linked polyvinyl alcohol), polypropylene, chitosan, ion exchange resin, hydrophobic adsorption resin, chelate resin, and synthetic adsorption resin. Among these, the ion exchange resin is preferably a phenol formaldehyde, polystyrene, acrylamide, or divinylbenzene ion exchange resin. Specific examples of the ion exchange resin include Duolite (Rohm and Haas), Amberlite (Rohm and Haas), Diaion (Mitsubishi Chemical), and Chitopearl (Fujibo Holdings). However, it is not limited to these.

  As will be described later, when a tag sequence is added to the asbestos-binding protein, it is preferable to select the carrier according to each tag sequence. That is, it is preferable to use a carrier on which a known substance known to bind to the tag sequence is immobilized. For example, when His-tag is used as the tag sequence, it is preferable that a metal ion is immobilized on the carrier. Specifically, the carrier is Ni Sepharose (manufactured by GE Healthcare Bioscience Co., Ltd.). Etc.). When FLAG, HA or c-myc is used as the tag sequence, it is preferable that an antibody capable of recognizing each tag sequence is immobilized on the carrier. When glutathione-S-transferase is used as the tag sequence, it is preferable that glutathione is immobilized on the carrier. When maltose binding protein is used as a tag sequence, it is preferable that maltose is immobilized on the carrier. According to the above configuration, the asbestos-binding protein can be securely and firmly supported on the carrier.

  In the asbestos adsorbent of the present embodiment, it is preferable that a layer for adjusting the humidity of the surface of the carrier is provided on the surface of the carrier. According to the above configuration, the humidity of the surface of the carrier can be increased. As a result, the asbestos adsorbent of the present embodiment is used under dry conditions (for example, in the gas phase, in the atmosphere, etc.) Even in such a case, the asbestos-binding protein supported on the carrier can exhibit activity. In other words, the asbestos adsorption effect of the asbestos adsorbent according to the present embodiment can be maintained over a long period of time.

  The specific configuration of the layer is not particularly limited. For example, the layer may be formed of a water-absorbing and hygroscopic polymer provided on the surface of the carrier, or a first functional group introduced into the carrier. preferable.

  The polymer is not particularly limited as long as it has a high moisture retention effect. For example, a polyacrylic acid polymer, a polyacrylate polymer, a polyamide polymer, a polyalcohol polymer, a polyether polymer, At least one selected from the group consisting of polyvinyl pyrrolidone polymers, polycarboxylic acid polymers, polyethyleneimine polymers, polyvinyl pyridine polymers, maleic acid polymers, polyvinyl phosphonic acids, polyvinyl sulfonic acids, polysaccharides, and polyamino acids. It is preferable that

  The method for providing the polymer on the surface of the carrier is not particularly limited. For example, the above polymer may be dissolved in a buffer and the carrier may be immersed in the solution. In addition, the carrier after being immersed in the solution can be appropriately dried.

  The first functional group is not particularly limited as long as it has a high effect of retaining moisture. For example, the first functional group is selected from the group consisting of a carboxyl group, a derivative of a carboxyl group, an amide group, an amino group, and a hydroxyl group. It is preferable that it is at least one.

  In addition, the method for introducing the first functional group into the carrier is not particularly limited, and the first functional group can be appropriately introduced using a known method according to the material of the carrier. For example, the functional group can be introduced onto the surface of the support by treating the support with an acid or alkali, or the functional group can be directly reacted with the substance having the functional group. It is possible to introduce a functional group onto the surface of the carrier, or to immobilize a substance having the functional group on the surface of the carrier with a known crosslinking agent to introduce the functional group onto the surface of the carrier. It is. In addition, although it does not specifically limit as a substance which has the said functional group, For example, it is preferable to use succinic anhydride, glutaric anhydride, or ethylenediamine.

  The asbestos-binding protein is not particularly limited as long as it is a protein that binds to asbestos, in other words, is an adsorbent protein. In the present specification, “asbestos-binding protein” may be referred to as “asbestos-adsorbing protein”. In the present specification, “asbestos binding” means, for example, a decrease in the number of asbestos fibers in an analysis using a phase contrast microscope, a scanning electron microscope, or a transmission electron microscope, or “asbestor” In the analysis using (manufactured by Silicon Bio Co., Ltd.), the determination can be made based on the fact that the concentration of the asbestos-containing solution is reduced.

  As described above, the specific configuration of the asbestos-binding protein is not particularly limited, and may be a protein derived from any organism such as a microorganism, a plant, or an animal. In the above organisms, the asbestos-binding protein is preferably derived from a microorganism.

  More specifically, the asbestos-binding protein is selected from the group consisting of OmpC, OmpA, DksA, HlpA, YgiW, H-NS, CspA-2, Cgl0974, TTC0984, GatZ, L1, L5, S1, S4 and S7. Preferably it is at least one protein selected. More preferably, the asbestos binding protein is at least one protein selected from the group consisting of OmpC, OmpA, DksA, HlpA, YgiW, H-NS, CspA-2, Cgl0974 and TTC0984, most preferably DksA.

The asbestos-binding protein may be a protein described in any of (I) to (III) below. That means
(I) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29, a protein comprising the amino acid sequence;
(II) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29. One or several amino acids A protein comprising an amino acid sequence in which residues are substituted, deleted, inserted and / or added, and having asbestos binding;
(III) SEQ ID NO: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29 has 55% or more homology with the amino acid sequence A protein comprising an amino acid sequence having an asbestos binding property.

  OmpA is a protein having a molecular weight of about 37 kD derived from E. coli K12. More specifically, OmpA is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (ACCESSION: NP_415477), and is a protein encoded by a gene (ompA) consisting of the base sequence shown in SEQ ID NO: 2.

  OmpC is a protein having a molecular weight of about 40 kD derived from E. coli K12. More specifically, OmpC is a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 (ACCESSION: NP — 416719), and is a protein encoded by a gene (ompC) consisting of the base sequence shown in SEQ ID NO: 4.

  HlpA is a protein having a molecular weight of about 17 kD derived from E. coli K12. More specifically, HlpA is a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 (ACCESSION: NP_414720), and is a protein encoded by the gene (hlpA) consisting of the base sequence shown in SEQ ID NO: 6.

  YgiW is a protein having a molecular weight of about 14 kD derived from E. coli K12. More specifically, YgiW is a protein consisting of the amino acid sequence shown in SEQ ID NO: 7 (ACCESSION: NP — 417496), and is a protein encoded by a gene (ygiW) consisting of the base sequence shown in SEQ ID NO: 8.

  DksA is a protein having a molecular weight of about 20 kD derived from E. coli K12. More specifically, DksA is a protein consisting of the amino acid sequence shown in SEQ ID NO: 9 (ACCESSION: NP_414687), and is a protein encoded by a gene (dksA) consisting of the base sequence shown in SEQ ID NO: 10.

  H-NS is a protein having a molecular weight of about 15 kD derived from E. coli K12. More specifically, H-NS is a protein consisting of the amino acid sequence shown in SEQ ID NO: 11 (ACCESSION: NP_415753), and is a protein encoded by a gene (hns) consisting of the base sequence shown in SEQ ID NO: 12. is there.

  CspA-2 is a protein having a molecular weight of about 5 kD derived from Pseudomonas putida KT2440. More specifically, CspA-2 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 13 (ACCESSION: NP_744611), and is encoded by a gene (cspA-2) consisting of the base sequence shown in SEQ ID NO: 14. It is a protein.

  Cgl0974 is a protein having a molecular weight of about 50 kD derived from Corynebacterium glutamicum ATCC13032. More specifically, Cgl0974 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 15 (ACCESSION: NP_600201), and is a protein encoded by a gene consisting of the base sequence shown in SEQ ID NO: 16 (cgl0974).

  TTC0984 is a protein having a molecular weight of about 10 kD derived from the hyperthermophilic bacterium HB27. More specifically, TTC0984 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 17 (ACCESSION: YP_004953), and is a protein encoded by a gene consisting of the base sequence shown in SEQ ID NO: 18 (ttc0984).

  GatZ is a protein known as tagatose 6-phosphate kinase derived from Escherichia coli K12 (ATCC 700926). GatZ is a protein consisting of the amino acid sequence shown in SEQ ID NO: 19 (ACCESSION: U00096 (Escherichia coli str. K-12 substr. MG1655, complete genome), Protein ID: AAC75156.1), and shown in SEQ ID NO: 20. It is a protein encoded by a gene consisting of a base sequence. GatZ can bind to all of chrysotile, amosite, and crocidolite, and tremolite, anthophyllite, and actinolite.

  L1 is a protein known as a ribosomal protein derived from Escherichia coli K12 (ATCC 700926). L1 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 21 (ACCESSION NUMBER: U00096 (Escherichia coli str. K-12 substr. MG1655, complete genome), Protein ID: AAC76958.1) and shown in SEQ ID NO: 22. It is a protein encoded by a gene consisting of a base sequence. L1 has particularly high binding activity to amosite and crocidolite.

  L5 is a protein known as a ribosomal protein derived from Escherichia coli K12 (ATCC 700926). L5 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 23 (ACCESSION NUMBER: U00096 (Escherichia coli str. K-12 substr. MG1655, complete genome), Protein ID: AAC76333.1) and shown in SEQ ID NO: 24 It is a protein encoded by a gene consisting of a base sequence. L5 has particularly high binding activity to amosite and crocidolite.

  S1 is a protein known as a ribosomal protein derived from Escherichia coli K12 (ATCC 700926). S1 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 25 (ACCESSION NUMBER: U00096 (Escherichia coli str. K-12 substr. MG1655, complete genome), Protein ID: AAC73997.1), and shown in SEQ ID NO: 26 It is a protein encoded by a gene consisting of a base sequence. S1 can bind to all of chrysotile, amosite, and crocidolite.

  S4 is a protein known as a ribosomal protein derived from Escherichia coli K12 (ATCC 700926). S4 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 27 (ACCESSION NUMBER: U00096 (Escherichia coli str. K-12 substr. MG1655, complete genome), Protein ID: AAC76321.1), and shown in SEQ ID NO: 28. It is a protein encoded by a gene consisting of a base sequence. S4 has particularly high binding activity to amosite and crocidolite.

  S7 is a protein known as a ribosomal protein derived from Escherichia coli K12 (ATCC 700926). S7 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 29 (ACCESSION NUMBER: U00096 (Escherichia coli str. K-12 substr. MG1655, complete genome), Protein ID: AAC76366.1), and shown in SEQ ID NO: 30 It is a protein encoded by a gene consisting of a base sequence. S7 has particularly high binding activity to amosite and crocidolite.

  As described above, the asbestos-binding protein is an amino acid described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29. In the sequence, it may be a protein comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and having asbestos binding. At this time, the site where one or several amino acids are substituted, deleted, inserted and / or added is the position of the protein after substitution, deletion, insertion and / or addition if the protein has asbestos binding. Any site in the amino acid sequence may be used. Here, “1 or several amino acid residues” is specifically the number of amino acid residues in the range of 10 or less, preferably the amino acid residue in the range of 6 or less.

  The above “having asbestos binding” is intended to prevent the binding of protein and asbestos from being dissociated in a solution containing sodium chloride having a concentration of 0.1 M (preferably 1 M). More specifically, a 25 mM Tris-HCl buffer (pH 7. 5) containing 0.1 M (preferably 1 M) sodium chloride and 0.5% polyoxyethylene sorbitan monolaurate (trade name: Tween 20 (registered trademark)). 5 or pH 8.0), it is intended that the binding of protein and asbestos does not dissociate.

  Proteins with high sequence identity to the amino acid sequences of OmpC, OmpA, DksA, HlpA, YgiW, H-NS, CspA-2, Cgl0974, TTC0984, GatZ, L1, L5, S1, S4 and S7 are also asbestos binding It can be used as a protein. In this case, the sequence identity is preferably 55% or more, more preferably 70% or more, and further preferably 80% or more. Preferably, it is more preferably 85% or more of sequence identity, more preferably 90% or more of sequence identity, and most preferably 95% or more of sequence identity. Information on the specific amino acid sequence and base sequence of the asbestos-binding protein can be obtained through a database such as NCBI.

  In the present invention, “homology” intends homology between two base sequences or two amino acid sequences. Homology is determined by comparing two sequences that are optimally aligned over the entire region of the sequence being compared. Here, in order to align the base sequence or amino acid sequence to be compared with each other in an optimal state, addition or deletion (for example, a gap) may be allowed. Such sequence homologies include, for example, FASTA (Pearson & Lipman, 1988, PNAS, 4, 2444-2448), BLAST (Altschul et al, 1990, Journal of Molecular Biol., 215, 403-410), CLUSTAL W (Thompson et al, 1994, Nucleic Acid Research, 22, 4673-4680).

  The above program is, for example, the WEB of an international DNA data bank operated in the DNA Data Bank of Japan (Center for Information Biology and DNA Data Bank of Japan; CIB / DDBJ). It is generally available on the page (http://www.ddbj.nig.ac.jp/Welcome-e.html). In addition, sequence homology can be determined using commercially available sequence analysis software. Specifically, the sequence homology is determined by DNASYS Pro ver. It can be calculated by performing homology analysis and aligning using the Smith-Waterman program of 2.06 (Hitachi Software Engineering).

  As the asbestos-binding protein, it is preferable to use a protein with a tag sequence added. In the present specification, “tag sequence” means an amino acid sequence artificially added to an asbestos-binding protein.

  Although the specific structure of the tag sequence is not particularly limited, for example, His-tag, FLAG, HA, glutathione-S-transferase (GST), c-myc, or maltose binding protein (MBP) is preferably used. . According to the above configuration, an asbestos adsorbent can be more easily produced by using a carrier that can specifically bind to a tag sequence. In addition, the position in the asbestos-binding protein to which the tag sequence is added is not particularly limited. For example, a tag sequence can be added to the N-terminus of the asbestos-binding protein, or a tag sequence can be added to the C-terminus of the asbestos-binding protein.

  In the asbestos adsorbent of the present embodiment, the asbestos-binding protein is supported on the carrier. At this time, the specific method for carrying is not particularly limited. For example, after asbestos-binding protein is dissolved in a buffer, it can be supported by immersing the carrier in the solution.

  Moreover, in the asbestos adsorbent of the present embodiment, it is preferable that the asbestos-binding protein is immobilized on the carrier via the second functional group. In addition, although the structure in which the said 2nd functional group is provided is not specifically limited, For example, it is preferable that the said 2nd functional group is provided on the surface of the said support | carrier. According to the above configuration, various bonds such as a covalent bond, a hydrophobic bond, an ionic bond, or a hydrogen bond are formed between the second functional group and the carrier, whereby the asbestos-binding protein is It can be supported more firmly on the carrier.

  Among the above bonds, a covalent bond is more preferable. If it is the said structure, since a support | carrier and an asbestos-binding protein are couple | bonded more firmly, it can prevent more reliably that an asbestos-binding protein falls from on a support | carrier. As a result, if it is the said structure, the removal effect of asbestos can be improved. Moreover, if it is the said structure, the asbestos adsorbent of this Embodiment can be used in various environments (for example, in the high salt concentration aqueous solution, an acidic solution, an alkaline solution, etc.). Moreover, if it is the said structure, when the asbestos adsorbent of this Embodiment becomes dirty or the adsorption effect falls, an asbestos adsorbent can be easily wash | cleaned using various solutions. Furthermore, with the above configuration, it is possible to prevent the asbestos-binding protein from falling off from the carrier even if a strong cleaning solution such as a surfactant, an acidic solution, or an alkaline solution is used during cleaning. The asbestos adsorption effect can be recovered.

  Although it does not specifically limit as said 2nd functional group, For example, it selects from the group which consists of an aldehyde group, an amino group, a carboxyl group, a hydroxyl group, a hydrazide group, a carbodiimide group, an epoxy group, a thiol group, an anhydrous ring, and a maleimide group Preferably, the functional group is at least one functional group.

  The second functional group and the first functional group are preferably the same functional group, or the carrier is preferably coated with the water-absorbing / hygroscopic polymer having the second functional group. . According to the said structure, the preparation process of an asbestos adsorbent can be simplified.

  In addition, the method for introducing the second functional group into the carrier is not particularly limited, and can be appropriately introduced using a known method according to the material of the carrier. For example, the functional group can be introduced onto the surface of the support by treating the support with an acid or alkali, or the functional group can be directly reacted with the substance having the functional group. It is possible to introduce a functional group onto the surface of the carrier, or to immobilize a substance having the functional group on the surface of the carrier with a known crosslinking agent to introduce the functional group onto the surface of the carrier. It is. In addition, although it does not specifically limit as a substance which has the said functional group, For example, it is preferable to use ethylenediamine, glutaraldehyde, or hydroxymethylmaleimide.

  The asbestos-binding protein is preferably immobilized on a carrier via a linker. According to the said structure, the freedom degree of arrangement | positioning of asbestos binding protein on a support | carrier becomes high. And it becomes easy for asbestos to approach an asbestos binding protein, As a result, the effect which removes asbestos can be heightened.

  The specific structure of the linker is not particularly limited. For example, maleimide, N-hydroxysuccinimidyl ester (NHS), imide ester, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (NHS, N-hydroxysuccinimidyl ester) EDC, 1-Ethyl-3- [3-dimethylaminopropyl] carbodiimido), and N- (p-maleimidophenyl) isocyanate (PMPI, N- [p-maleimidophenyl] isocynate), nitrilotriacetic acid (NTA, nitrilotriacetone) And at least one compound selected from the group consisting of maltose.

The asbestos-binding protein is preferably immobilized on a carrier via a metal ion. At this time, the type of the metal ion is not particularly limited, but for example, Ni ²⁺ , Ca ²⁺ , Mg ²⁺ , or Zn ²⁺ is preferably used. Moreover, when using His-tag as mentioned above, it is more preferable to use Ni ^<2+> among these metal ions. According to the above configuration, the asbestos-binding protein can be easily immobilized on the carrier.

  Hereinafter, specific examples of a specific method for supporting an asbestos-binding protein on a carrier will be described, but the present invention is not limited thereto.

  For example, after the carrier is silanized with γ-aminopropyltriethoxysilane or the like, an aldehyde group is introduced onto the surface of the carrier with glutaraldehyde or the like. Then, the asbestos-binding protein can be supported on the carrier by binding the aldehyde group and the asbestos-binding protein.

  In addition, an amino group is introduced on the surface of the carrier by immersing the carrier in an ethylenediamine solution, and the asbestos-binding protein is supported on the carrier by binding the amino group and the asbestos-binding protein. Can do.

  Further, the asbestos-binding protein can be supported on the carrier by immersing the untreated carrier (a carrier into which no functional group has been artificially introduced) in a solution containing the asbestos-binding protein.

  Moreover, the carrier is coated with a polymer having a specific functional group, and the asbestos-binding protein can be supported on the carrier by binding the functional group and the asbestos-binding protein.

  In addition, asbestos-binding protein is mixed on a material for producing a carrier as a powder or liquid, and then the carrier is produced according to a conventional method using the material, thereby asbestos-binding protein on the carrier. Can be supported.

  Examples of asbestos that can be adsorbed by the asbestos adsorbent of the present embodiment include chrysotile, crocidolite, amosite, anthophyllite, tremolite, and actinolite. Moreover, since the asbestos adsorbent of this embodiment has high binding specificity to chrysotile, crocidolite, and amosite, it is preferably used as a chrysotile adsorbent, a crocidolite adsorbent, or an amosite adsorbent. Moreover, since the asbestos adsorbent carrying DksA shown in the following examples has the highest binding specificity to chrysotile, it is most preferably used as the chrysotile adsorbent.

  Further, the asbestos adsorbent according to the present embodiment can be produced by combining two or more carriers. For example, a column carrier carrying asbestos binding protein can be immobilized on a nonwoven fabric or the like.

  Moreover, if the asbestos adsorption material of this Embodiment is used, the effective asbestos removal method can be provided.

  For example, the asbestos removal method includes at least one of a discharge step of spraying the asbestos adsorbent of the present invention on the asbestos removal work site before work and a spraying step of spraying the asbestos adsorbent of the present invention in the work space before and after the work. It is preferable that it is the method of including. According to the above configuration, scattering and re-scattering of asbestos inside and outside the work space can be effectively prevented.

  Although it does not specifically limit as a target (asbestos removal work location) which sprays an asbestos adsorbent in the said discharge process, For example, it may be a material (for example, building materials etc.) containing asbestos, a wall, a floor, or a ceiling of a work room. preferable.

  Moreover, it is preferable that the asbestos adsorbent used in the discharging step is appropriately dissolved in a known buffer or the like. According to the said structure, discharge of asbestos adsorbent can be made easy. The buffer preferably contains a surfactant. According to the said structure, scattering of asbestos can be prevented more reliably.

  Moreover, the specific means for discharging the asbestos adsorbent in the discharge step is not particularly limited, and a known discharge means can be used as appropriate.

  Although the target which sprays asbestos adsorbent in the said spraying process is not specifically limited, For example, it is preferable that it is in the air | atmosphere of the working space which is performing the asbestos removal operation | work. According to the said structure, the sprayed asbestos adsorption material can adsorb | suck to the asbestos scattered in air | atmosphere. Then, the asbestos adsorbent adsorbed on the asbestos settles on the floor of the work space while the asbestos is adsorbed. As a result, asbestos scattered in the atmosphere can be removed.

  It is preferable that the asbestos adsorbent used in the spraying process is appropriately dissolved in a known buffer or the like. According to the said structure, spraying of an asbestos adsorbent can be made easy. The buffer preferably contains a surfactant. According to the said structure, scattering of asbestos can be prevented.

  Moreover, the specific means for spraying the asbestos adsorbent in the spraying step is not particularly limited, and a known spraying means can be used as appropriate.

  Examples of the present invention will be described below, but the present invention is not limited thereto. In addition, DksA is mentioned and demonstrated as an example of a protein couple | bonded with asbestos below.

[Example 1]
Asbestos-binding protein (DksA) was prepared by the following procedure. That is, the DksA gene (SEQ ID NO: 10) was inserted into the expression vector pET21-b (manufactured by Novagen), and Escherichia coli MV1184 strain was transformed with the expression vector. Note that a His tag can be linked to DksA by using pET21-b. An expression vector into which the target DNA fragment was inserted was extracted from the obtained colony and named pET DksA.

  Escherichia coli Rosetta BL21 (DE3) pLyS (manufactured by Novagen) transformed with pET DksA was cultured overnight at 37 ° C. in 2 × YT medium. After overnight culture, 1% of the culture solution was inoculated against fresh 2 × YT medium and further cultured at 37 ° C.

After culturing until OD ₆₀₀ reaches 0.4, isopropyl-β-D-cytogalactopyranoside is added to the culture solution so that the final concentration is 0.5 mM, and the culture is further continued for 7 hours. went.

  The culture solution after the culture was centrifuged, and E. coli was recovered as a pellet. A buffer (25 mM Tris-HCl, pH 7.5, 0.1% Tween 20 (registered trademark)) was added to the above pellet to suspend it, and Escherichia coli was disrupted by ultrasonication. After crushing, the crushing solution was centrifuged at 20,000 × g for 15 minutes. After centrifugation, the supernatant containing DksA was collected. Hereinafter, the supernatant is referred to as “DksA cell lysate”.

  DksA was further purified from the DksA cell disruption solution. The method will be described below.

  Purification of DksA from the above DksA cell disruption solution was performed using 5 mL of HisTrap FF (manufactured by GE Healthcare). That is, a column (HisTrap FF) was equilibrated with a buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1% Tween 20 (registered trademark), 10% glycerol), and the DksA cell disruption solution was applied to the column. Was added. Next, after washing the column with the buffer containing 5 mM imidazole, DksA was eluted from the column with the buffer containing 500 mM imidazole.

  The purity of DksA was then confirmed by polyacrylamide gel electrophoresis. The result is shown in FIG. As a result of the purification step, 95% or more of the protein in the eluate was DksA. In other words, DksA was successfully obtained with high purity by the above purification step.

[Example 2]
Below, an example of the method of carrying | supporting asbestos binding protein on a support | carrier is demonstrated.

As the carrier, a nonwoven fabric (100% polyester, basis weight 135 g / m ² ) was used. The nonwoven fabric was immersed in a buffer solution (10 mM Tris-HCl, pH 7.5, 10 mM NaCl) overnight, and then immersed in a solution containing asbestos-binding protein (DksA) at 20 μg / ml for 1 hour. . Next, the nonwoven fabric was washed with the buffer solution. As described above, an asbestos adsorbent was prepared by supporting an asbestos-binding protein on a carrier.

Example 3
The asbestos adsorption capacity of the asbestos adsorbent produced in Example 2 was measured. The method will be described below.

  First, the asbestos adsorbent produced in Example 2 was immersed in an asbestos solution containing asbestos at 100 μg / ml. Then, after 1 hour, the concentration of asbestos in the asbestos solution was measured.

  The concentration of asbestos in the asbestos solution was measured according to the following procedure. That is, the asbestos solution was centrifuged at 13,000 g for 10 minutes to precipitate the chrysotile in the asbestos solution, and the supernatant was removed. Then, an enzyme solution of an asbestos detection kit “Asbestor” (manufactured by Silicon Bio Inc.) was added to the precipitate. After reacting the enzyme with each sample, washing treatment was performed to remove excess enzyme. In addition, these specific operation followed the protocol attached to the said kit. After washing and centrifugation, the supernatant was removed, and 100 μl of buffer (25 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.5% Tween 20 (registered trademark)) was added to the pellet. It was. This again dispersed the pellets (chrysotile).

  10 μL of the above dispersion was taken in a 96-well plate, and 100 μL of a luminescent substrate CDP-Star Detection Reagent (manufactured by GE Healthcare) was added to the dispersion. After the reaction for 10 minutes, the luminescence value was measured using a multi-label measuring device (wallac ARVO SX).

  The amount of asbestos adsorbed on the asbestos adsorbent was calculated by measuring the amount of asbestos remaining in the asbestos solution as described above.

  As a comparative example, an asbestos adsorbent prepared using “ultra pure water” instead of “a solution containing asbestos-binding protein (DksA) at 20 μg / ml” in Example 2 was used. In addition, the asbestos adsorption ability of the asbestos adsorbent was also measured for the asbestos adsorbent of the comparative example according to the method of Example 3 described above.

  Table 1 shows the results of measuring the asbestos adsorption capacity of the asbestos adsorbent of this example and the asbestos adsorption capacity of the asbestos adsorbent of the comparative example.

  As shown in Table 1, it was revealed that the non-woven fabric carrying asbestos-binding protein has a higher asbestos adsorption capacity than the non-loaded non-woven fabric.

Example 4
Asbestos adsorbent was prepared using Ni Sepharose 6 Fast Flow (manufactured by GE Healthcare Biosciences) as the carrier.

  Specifically, 40 μg of asbestos-binding protein (prepared in Example 1) was immobilized on 0.2 ml of Ni Sepharose 6 Fast Flow. The specific immobilization method followed the protocol attached to the carrier.

  The asbestos adsorbent produced as described above was immersed in an asbestos solution containing asbestos at 20 μg / ml. Then, after 1 hour, the concentration of asbestos in the asbestos solution was measured. The asbestos concentration in the asbestos solution was measured by the same method as in Example 3. As a comparative example, Ni Sepharose 6 Fast Flow not supporting DksA was used.

  Table 2 shows the results of measuring the asbestos adsorption capacity of the asbestos adsorbent of this example and the asbestos adsorption capacity of the asbestos adsorbent of the comparative example. In Table 2, “DksA +” indicates Ni Sepharose 6 Fast Flow supporting DksA, and “DksA−” indicates unsupported.

  As shown in Table 2, it was revealed that Ni Sepharose 6 Fast Flow loaded with asbestos-binding protein has higher asbestos adsorption ability than Ni Sepharose 6 Fast Flow not loaded.

Example 5
An asbestos adsorption filter was produced using a non-woven fabric (100% polyester, basis weight 135 g / m ² ). The method is described below.

  The nonwoven fabric was immersed in a coating solution (5% polyacrylic acid, 0.5% polyethylene glycol 400 diglycidyl ether), washed with water, and dried at 85 ° C. Thereafter, the nonwoven fabric was immersed in a 0.2% aqueous sodium hydroxide solution at 60 ° C. for 2 hours, washed with water and dried at 85 ° C. to obtain a nonwoven fabric coated with polyacrylic acid.

Next, the coated nonwoven fabric was immersed in a PBS solution containing 0.2M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 0.05M N-hydroxysuccinimide for 1 hour. Thereafter, the nonwoven fabric was immersed in a 3 mM N- (5-amino-1-carboxypentyl) iminodiacetic acid solution (AB-NTA solution) for 2 hours, washed with water, and then immersed in a 1M 2-aminoethanol solution (pH 8.5). . Next, the nonwoven fabric was immersed in a 2.5 mM nickel chloride solution (NiCl ₂ solution) for 10 minutes, washed with water and air-dried to obtain a nonwoven fabric in which Ni-NTA was introduced into the carboxyl group of polyacrylic acid.

  Finally, the Ni-NTA-introduced non-woven fabric is immersed in a solution containing 30 μg / ml of the asbestos-binding protein (DksA) for 1 hour to utilize the binding property of the His tag to nickel (Ni). -DksA was immobilized on the NTA-introduced nonwoven fabric. Subsequently, the nonwoven fabric was washed with water and then dehydrated by centrifugation (3000 rpm for 10 minutes) to obtain an asbestos adsorption filter.

Example 6
The asbestos adsorption ability in the gas phase of the filter produced in Example 5 was measured. The method is described below.

  About 0.4 g of chrysotile standard sample (JAWE111, Japan Work Environment Measurement Association) was put into an antistatic bag. Next, the asbestos adsorption filter prepared in Example 5 was placed on the upper stage of the two-stage collection holder (EMO-47, two-stage adapter for EMO-47, both manufactured by GL Science), and the membrane filter (pore diameter 0.8 μm, A 47 mm diameter, manufactured by Advantech) was attached, and a two-stage collection holder was placed in the antistatic bag. Thereafter, the chrysotile is dusted in the antistatic bag and allowed to stand for 2 minutes, and then the air 5 L in the antistatic bag is circulated in the two-stage collection holder at a flow rate of 1.9 L / min. Atmospheric chrysotile was collected by a two-stage collection holder. After the collection, the chrysotile on the membrane filter arranged in the lower stage was observed with a phase contrast microscope by the acetone-triacetin method, and the number of fibers in a 300 μm diameter frame was counted in 10 fields. That is, the number of fibers that passed through the asbestos adsorption filter without being adsorbed by the asbestos adsorption filter arranged in the upper stage and then captured by the membrane filter arranged in the lower stage was counted in 10 fields. In addition, as a comparative example at this time, a Ni-NTA nonwoven fabric not supporting DksA was used.

  In Table 3, the result of having measured the asbestos adsorption ability of the asbestos adsorption filter of a present Example and the Ni-NTA nonwoven fabric of a comparative example is shown. In addition, the air target fiber in Table 3 indicates a fiber having a fiber width of less than 3 μm, a length of 5 μm or more and an aspect ratio of 1: 3 or more according to the air analysis standard. Indicates a non-applicable fiber.

  As shown in Table 3, the number of target air fibers in the example was smaller than that in the comparative example. Further, in this example, only the chrysotile standard sample is put in the antistatic bag, so it is considered that the non-target fibers are almost asbestos. From these facts, the filter of the example adsorbed more asbestos fibers than the filter of the comparative example. In other words, it has been clarified that the asbestos adsorption filter carrying the asbestos-binding protein has a higher asbestos adsorption ability even in the gas phase as compared with the filter not carrying it.

  Note that the present invention is not limited to the configurations described above, and various modifications are possible within the scope of the claims, and technical means disclosed in different embodiments and examples respectively. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention.

  The present invention can strongly adsorb asbestos and prevent once adsorbed asbestos from splashing again, so that an asbestos removal sheet, an asbestos removal filter, an asbestos protective clothing, a respirator (for example, a mask) ) Can be used. Each of these can be used for a liquid phase or a gas phase. Of course, it can be used for both liquid phase and gas phase.

Claims (13)

A carrier;
And an asbestos-binding protein supported on the carrier.   The asbestos-binding protein is at least one selected from the group consisting of OmpC, OmpA, DksA, HlpA, YgiW, H-NS, CspA-2, Cgl0974, TTC0984, GatZ, L1, L5, S1, S4 and S7. The asbestos adsorbent according to claim 1, which is a protein. The asbestos-adsorbing material according to claim 1, wherein the asbestos-binding protein is a protein according to any one of the following (I) to (III).
(I) A protein comprising the amino acid sequence set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29.
(II) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29. One or several amino acids A protein comprising an amino acid sequence in which residues are substituted, deleted, inserted and / or added, and which has asbestos binding.
(III) SEQ ID NO: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29 has 55% or more homology with the amino acid sequence A protein comprising an amino acid sequence having an asbestos binding property.   The shape of the carrier is cotton, filter paper, sheet, honeycomb, granule, powder, mesh, porous, or gel, according to any one of claims 1 to 3. Asbestos adsorbent as described in 1.   5. The asbestos adsorbent according to claim 1, wherein a layer for adjusting the humidity of the surface of the carrier is provided on the surface of the carrier.   6. The layer according to claim 5, wherein the layer is formed by a water-absorbing and hygroscopic polymer provided on the surface of the carrier, or a first functional group introduced on the surface of the carrier. Asbestos adsorbent.   The polymer is a polyacrylic acid polymer, polyacrylate polymer, polyamide polymer, polyalcohol polymer, polyether polymer, polyvinyl pyrrolidone polymer, polycarboxylic acid polymer, polyethyleneimine polymer, polyvinyl pyridine polymer. The asbestos adsorbent according to claim 6, wherein the adsorbent is at least one selected from the group consisting of a polymer, a maleic acid polymer, polyvinyl phosphonic acid, polyvinyl sulfonic acid, a polysaccharide, and a polyamino acid.   The asbestos adsorption according to claim 6, wherein the first functional group is at least one selected from the group consisting of a carboxyl group, a derivative of a carboxyl group, an amide group, an amino group, and a hydroxyl group. Wood.   The asbestos adsorbent according to any one of claims 1 to 8, wherein the asbestos-binding protein is immobilized on the carrier via a second functional group, a metal ion, or a linker. .   The second functional group is at least one functional group selected from the group consisting of an aldehyde group, amino group, carboxyl group, hydroxyl group, hydrazide group, carbodiimide group, epoxy group, thiol group, anhydrous ring, and maleimide group. The asbestos adsorbent according to claim 9, wherein the adsorbent is asbestos.   The linker is maleimide, N-hydroxysuccinimidyl ester, imide ester, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N- (p-maleimidophenyl) isocyanate, nitrilotriacetic acid, glutathione, and maltose The asbestos adsorbent according to claim 9, which is at least one compound selected from the group consisting of:   The asbestos adsorbent according to any one of claims 1 to 11, which adsorbs asbestos in a gas phase.   An asbestos removal method using the asbestos adsorbent according to any one of claims 1 to 12.

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