JP2019503685A - 組換え微生物を利用した金属ナノ粒子及び金属硫化物ナノ粒子の製造方法 - Google Patents
組換え微生物を利用した金属ナノ粒子及び金属硫化物ナノ粒子の製造方法 Download PDFInfo
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- JP2019503685A JP2019503685A JP2018535133A JP2018535133A JP2019503685A JP 2019503685 A JP2019503685 A JP 2019503685A JP 2018535133 A JP2018535133 A JP 2018535133A JP 2018535133 A JP2018535133 A JP 2018535133A JP 2019503685 A JP2019503685 A JP 2019503685A
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- nanoparticles
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- sulfide
- hydroxide
- metal nanoparticles
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- 239000002082 metal nanoparticle Substances 0.000 title claims abstract description 113
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 105
- 244000005700 microbiome Species 0.000 title claims abstract description 77
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 45
- 229910052976 metal sulfide Inorganic materials 0.000 title claims abstract description 37
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 44
- 108090000157 Metallothionein Proteins 0.000 claims abstract description 35
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 claims abstract description 34
- 102000003792 Metallothionein Human genes 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 34
- 229910052751 metal Inorganic materials 0.000 claims abstract description 20
- 239000002184 metal Substances 0.000 claims abstract description 17
- 229910052717 sulfur Inorganic materials 0.000 claims abstract description 10
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000011593 sulfur Substances 0.000 claims abstract description 9
- 239000002245 particle Substances 0.000 claims abstract description 8
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 61
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 52
- 239000004332 silver Substances 0.000 claims description 52
- 229910052709 silver Inorganic materials 0.000 claims description 51
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 49
- 239000011572 manganese Substances 0.000 claims description 35
- SITVSCPRJNYAGV-UHFFFAOYSA-L tellurite Chemical compound [O-][Te]([O-])=O SITVSCPRJNYAGV-UHFFFAOYSA-L 0.000 claims description 31
- 239000002131 composite material Substances 0.000 claims description 28
- 239000011669 selenium Substances 0.000 claims description 28
- 239000010949 copper Substances 0.000 claims description 27
- 239000011651 chromium Substances 0.000 claims description 26
- 238000012258 culturing Methods 0.000 claims description 25
- 239000010931 gold Substances 0.000 claims description 25
- 229910052684 Cerium Inorganic materials 0.000 claims description 24
- 229910052777 Praseodymium Inorganic materials 0.000 claims description 24
- 229910052746 lanthanum Inorganic materials 0.000 claims description 24
- FZLIPJUXYLNCLC-UHFFFAOYSA-N lanthanum atom Chemical compound [La] FZLIPJUXYLNCLC-UHFFFAOYSA-N 0.000 claims description 23
- 229910052793 cadmium Inorganic materials 0.000 claims description 22
- GWXLDORMOJMVQZ-UHFFFAOYSA-N cerium Chemical compound [Ce] GWXLDORMOJMVQZ-UHFFFAOYSA-N 0.000 claims description 22
- 229910052738 indium Inorganic materials 0.000 claims description 22
- APFVFJFRJDLVQX-UHFFFAOYSA-N indium atom Chemical compound [In] APFVFJFRJDLVQX-UHFFFAOYSA-N 0.000 claims description 22
- 229910052759 nickel Inorganic materials 0.000 claims description 22
- PUDIUYLPXJFUGB-UHFFFAOYSA-N praseodymium atom Chemical compound [Pr] PUDIUYLPXJFUGB-UHFFFAOYSA-N 0.000 claims description 22
- 229910052772 Samarium Inorganic materials 0.000 claims description 21
- 229910021645 metal ion Inorganic materials 0.000 claims description 21
- 229910052688 Gadolinium Inorganic materials 0.000 claims description 20
- 229910052779 Neodymium Inorganic materials 0.000 claims description 20
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims description 20
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 claims description 20
- 239000010941 cobalt Substances 0.000 claims description 20
- 229910017052 cobalt Inorganic materials 0.000 claims description 20
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims description 20
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 claims description 18
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 claims description 18
- 229910052788 barium Inorganic materials 0.000 claims description 18
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 claims description 18
- 229910052748 manganese Inorganic materials 0.000 claims description 18
- 229910052750 molybdenum Inorganic materials 0.000 claims description 18
- 239000011733 molybdenum Substances 0.000 claims description 18
- 229910052693 Europium Inorganic materials 0.000 claims description 17
- KZUNJOHGWZRPMI-UHFFFAOYSA-N samarium atom Chemical compound [Sm] KZUNJOHGWZRPMI-UHFFFAOYSA-N 0.000 claims description 17
- 229910052714 tellurium Inorganic materials 0.000 claims description 17
- PORWMNRCUJJQNO-UHFFFAOYSA-N tellurium atom Chemical compound [Te] PORWMNRCUJJQNO-UHFFFAOYSA-N 0.000 claims description 17
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 claims description 16
- QEFYFXOXNSNQGX-UHFFFAOYSA-N neodymium atom Chemical compound [Nd] QEFYFXOXNSNQGX-UHFFFAOYSA-N 0.000 claims description 16
- VSZWPYCFIRKVQL-UHFFFAOYSA-N selanylidenegallium;selenium Chemical compound [Se].[Se]=[Ga].[Se]=[Ga] VSZWPYCFIRKVQL-UHFFFAOYSA-N 0.000 claims description 16
- 229910003321 CoFe Inorganic materials 0.000 claims description 15
- 229910001030 Iron–nickel alloy Inorganic materials 0.000 claims description 15
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims description 15
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 claims description 15
- 229910052711 selenium Inorganic materials 0.000 claims description 15
- JBQYATWDVHIOAR-UHFFFAOYSA-N tellanylidenegermanium Chemical compound [Te]=[Ge] JBQYATWDVHIOAR-UHFFFAOYSA-N 0.000 claims description 15
- SZKTYYIADWRVSA-UHFFFAOYSA-N zinc manganese(2+) oxygen(2-) Chemical compound [O--].[O--].[Mn++].[Zn++] SZKTYYIADWRVSA-UHFFFAOYSA-N 0.000 claims description 15
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 14
- 229910052802 copper Inorganic materials 0.000 claims description 14
- DLINORNFHVEIFE-UHFFFAOYSA-N hydrogen peroxide;zinc Chemical compound [Zn].OO DLINORNFHVEIFE-UHFFFAOYSA-N 0.000 claims description 14
- DMTIXTXDJGWVCO-UHFFFAOYSA-N iron(2+) nickel(2+) oxygen(2-) Chemical compound [O--].[O--].[Fe++].[Ni++] DMTIXTXDJGWVCO-UHFFFAOYSA-N 0.000 claims description 14
- 229940105296 zinc peroxide Drugs 0.000 claims description 14
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 claims description 13
- 229910052792 caesium Inorganic materials 0.000 claims description 13
- TVFDJXOCXUVLDH-UHFFFAOYSA-N caesium atom Chemical compound [Cs] TVFDJXOCXUVLDH-UHFFFAOYSA-N 0.000 claims description 13
- 229910052804 chromium Inorganic materials 0.000 claims description 13
- QRXDDLFGCDQOTA-UHFFFAOYSA-N cobalt(2+) iron(2+) oxygen(2-) Chemical compound [O-2].[Fe+2].[Co+2].[O-2] QRXDDLFGCDQOTA-UHFFFAOYSA-N 0.000 claims description 13
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 13
- 229910052737 gold Inorganic materials 0.000 claims description 13
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 claims description 13
- 229910052753 mercury Inorganic materials 0.000 claims description 13
- 229910052712 strontium Inorganic materials 0.000 claims description 12
- CIOAGBVUUVVLOB-UHFFFAOYSA-N strontium atom Chemical compound [Sr] CIOAGBVUUVVLOB-UHFFFAOYSA-N 0.000 claims description 12
- 229910052782 aluminium Inorganic materials 0.000 claims description 10
- AFNRRBXCCXDRPS-UHFFFAOYSA-N tin(ii) sulfide Chemical compound [Sn]=S AFNRRBXCCXDRPS-UHFFFAOYSA-N 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 9
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 8
- GKCNVZWZCYIBPR-UHFFFAOYSA-N sulfanylideneindium Chemical compound [In]=S GKCNVZWZCYIBPR-UHFFFAOYSA-N 0.000 claims description 8
- CADICXFYUNYKGD-UHFFFAOYSA-N sulfanylidenemanganese Chemical compound [Mn]=S CADICXFYUNYKGD-UHFFFAOYSA-N 0.000 claims description 8
- 229910052727 yttrium Inorganic materials 0.000 claims description 8
- VWQVUPCCIRVNHF-UHFFFAOYSA-N yttrium atom Chemical compound [Y] VWQVUPCCIRVNHF-UHFFFAOYSA-N 0.000 claims description 8
- HASDHSVWTCCGIM-UHFFFAOYSA-N zinc iron(2+) oxygen(2-) Chemical compound [O-2].[O-2].[Fe+2].[Zn+2] HASDHSVWTCCGIM-UHFFFAOYSA-N 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- AIBQNUOBCRIENU-UHFFFAOYSA-N nickel;dihydrate Chemical compound O.O.[Ni] AIBQNUOBCRIENU-UHFFFAOYSA-N 0.000 claims description 7
- RCYIWFITYHZCIW-UHFFFAOYSA-N 4-methoxybut-1-yne Chemical compound COCCC#C RCYIWFITYHZCIW-UHFFFAOYSA-N 0.000 claims description 6
- PLLZRTNVEXYBNA-UHFFFAOYSA-L cadmium hydroxide Chemical compound [OH-].[OH-].[Cd+2] PLLZRTNVEXYBNA-UHFFFAOYSA-L 0.000 claims description 6
- 229910000420 cerium oxide Inorganic materials 0.000 claims description 6
- CQQZFSZWNXAJQN-UHFFFAOYSA-K europium(3+);trihydroxide Chemical compound [OH-].[OH-].[OH-].[Eu+3] CQQZFSZWNXAJQN-UHFFFAOYSA-K 0.000 claims description 6
- CMGJQFHWVMDJKK-UHFFFAOYSA-N lanthanum;trihydrate Chemical compound O.O.O.[La] CMGJQFHWVMDJKK-UHFFFAOYSA-N 0.000 claims description 6
- BMMGVYCKOGBVEV-UHFFFAOYSA-N oxo(oxoceriooxy)cerium Chemical compound [Ce]=O.O=[Ce]=O BMMGVYCKOGBVEV-UHFFFAOYSA-N 0.000 claims description 6
- ZLGIGTLMMBTXIY-UHFFFAOYSA-K praseodymium(3+);trihydroxide Chemical compound [OH-].[OH-].[OH-].[Pr+3] ZLGIGTLMMBTXIY-UHFFFAOYSA-K 0.000 claims description 6
- 241000195493 Cryptophyta Species 0.000 claims description 5
- UBEWDCMIDFGDOO-UHFFFAOYSA-N cobalt(2+);cobalt(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Co+2].[Co+3].[Co+3] UBEWDCMIDFGDOO-UHFFFAOYSA-N 0.000 claims description 5
- ILCLBMDYDXDUJO-UHFFFAOYSA-K gadolinium(3+);trihydroxide Chemical compound [OH-].[OH-].[OH-].[Gd+3] ILCLBMDYDXDUJO-UHFFFAOYSA-K 0.000 claims description 5
- FSJWWSXPIWGYKC-UHFFFAOYSA-M silver;silver;sulfanide Chemical compound [SH-].[Ag].[Ag+] FSJWWSXPIWGYKC-UHFFFAOYSA-M 0.000 claims description 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 4
- MARUHZGHZWCEQU-UHFFFAOYSA-N 5-phenyl-2h-tetrazole Chemical compound C1=CC=CC=C1C1=NNN=N1 MARUHZGHZWCEQU-UHFFFAOYSA-N 0.000 claims description 4
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- 229910052984 zinc sulfide Inorganic materials 0.000 claims description 4
- WUPHOULIZUERAE-UHFFFAOYSA-N 3-(oxolan-2-yl)propanoic acid Chemical compound OC(=O)CCC1CCCO1 WUPHOULIZUERAE-UHFFFAOYSA-N 0.000 claims description 3
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- FQMNUIZEFUVPNU-UHFFFAOYSA-N cobalt iron Chemical compound [Fe].[Co].[Co] FQMNUIZEFUVPNU-UHFFFAOYSA-N 0.000 claims description 3
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- PFNQVRZLDWYSCW-UHFFFAOYSA-N (fluoren-9-ylideneamino) n-naphthalen-1-ylcarbamate Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1=NOC(=O)NC1=CC=CC2=CC=CC=C12 PFNQVRZLDWYSCW-UHFFFAOYSA-N 0.000 claims description 2
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Abstract
Description
[実施例]
以下、実施例を通じて本発明をより詳細に説明する。これらの実施例には単に本発明を例示するためのもので、本発明の範囲がこれらの実施例により制限されることと解釈されないことは当業界において通常の知識を有する者にとって自明である。したがって、本発明の実質的な範囲は添付された請求項とそれらの等価物によって定義されるということである。
1−1:メタロチオネイン発現ベクターの製作
シュードモナス プチダ(Pseudomonas putida)KT2440の染色体DNA(genomic DNA)を鋳型として、配列番号3と4のプライマーにポリメラーゼ連鎖反応(PCR)を随行して、メタロチオネイン (metallothionenin,MT)の塩基配列をコードするMT遺伝子の切片を製作した。
[配列番号3]:5’−ATAGAATTCATGAACGATCACCACCACCACAA−3’
[配列番号4]:5’−TATCTGCAGTTAGGGCGAGATCGGATCACTC−3’
シロイヌナズナ(Arabidopsis thalinana)のファイトケラチン合成酵素(phytochelatin synthase,PCS)を確保するために、シロイヌナズナの相補的染色体(cDNA)を鋳型として、ファイトケラチン合成酵素の塩基配列をコードするファイトケラチン合成酵素の遺伝子切片を製作した。上記ポリメラーゼ連鎖反応は下記配列番号5と6のプライマーを利用して遂行した。
[配列番号5]:5’− ATAGAATTCATGGCTATGGCGAGTTTATATCGG −3’
[配列番号6]:5’− TATGCATGCTTAATAGGCAGGAGCAGCGAGATC −3’
メタロチオネインとファイトケラチン合成酵素を同時に発現するベクターを製作するために、先に製作されたpYJ−MTとpYJ−PCSプラスミドからpYJ−MT−PCSを製作した。 pYJ−PCSベクターを下記配列番号7と8のプライマーを利用してポリメラーゼ連鎖反応(PCR)を随行した。
[配列番号7]:5’−ATACTGCAGTTGACAATTAATCATCGGCTCGTATA−3’
[配列番号8]:5’−TATGCATGCTTAATAGGCAGGAGCAGCGAGA−3’
メタロチオネインとファイトケラチン合成酵素を同時に発現するように実施例1−3で製作された組換えプラスミドのpYJ−MT−PCSを大腸菌DH5αに導入した。形質転換された組換え大腸菌をLB液体培地100mLの含まれた250mLフラスコに接種して37℃で培養した。上記組換え大腸菌の光学密度(Optical density,OD)が分光光度計600nm波長で0.6の数値を示す時IPTG(Isopropyl−D−1−thiogalactopyranoside)を添加してメタロチオネインとファイトケラチン合成酵素の発現を誘導した。pYJ−MT−PCSに挿入されているtacプロモーターはIPTGによって作動し、培養液のIPTGの最終濃度が1 mMになるように添加してその発現を誘導した。
2−1:組換え微生物を利用した単一金属ナノ粒子の合成
上記、実施例1で製造された組換え微生物でメタロチオネインとファイトケラチン合成酵素の発現をIPTGで誘導して1時間後に大腸菌が培養されているLB液体培地(Tryptone 10g/L,Yeast extract 5g/L,NaCl 5g/L,pH 6.5)にそれぞれバリウム(Ba,(CH3COO)2Ba,0.5mM)及びジルコニウム(Zr,K2ZrF6,0.5mM)、モリブデン(Mo,Na2MoO4・2H2O,0.5mM)、インジウム(In,InCl2・4H2O,0.5mM)、すず(Sn,SnCl2・4H2O,0.5mM)、ランタン(La,La(NO3)3・6H2O,0.5mM)、セリウム(Ce,Ce(NO3)3・6H2O,0.5mM)またはプラセオジム(Pr,Pr(NO3)3・6H2O,0.5mM)を添加し、12時間の間、追加で培養した。全体培養液を4℃、3500 rpmで15分間、遠心分離した後上層液を捨てて大腸菌ペレットを修得した。PBSのバッファーで大腸菌を洗浄する過程を三回繰り返した後、凍結乾燥機で真空状態で一日以上乾燥させた。ナノ粒子が合成されたことを透過電子顕微鏡(Transmission electron microscopy,TEM)(Tecnai F20,Philips,Netherlands)と、X線分光分析機(Energy−dispersive X−ray spectoscopy,EDX)(D/MAX−2,500,Rigaku,Japan)with CuKa radiation(λ=1.5406Å))を利用して調査した。
実施例2−1で製造された単一金属ナノ粒子の表面官能基を確認しようと、常温で赤外線分光器(Fourier−Transform Infrared Spectrophotometer,FT−IR)(Nicolet(登録商標)iS(登録商標)50,Thermo Scientific,USA)を利用して400〜4000cm−1の範囲で合成された単一金属ナノ粒子を分析した。
3−1:組換え微生物を利用した複合金属ナノ粒子の合成
実施例1で製造された組換え微生物でメタロチオネインとファイトケラチン合成酵素の発現をIPTGで誘導して1時間後に、1時間後に大腸菌が培養されているLB液体培地(Tryptone 10g/L,Yeast extract 5g/L,NaCl 5g/L,pH 6.5)にそれぞれi)コバルトと鉄(Co,CoCl2,0.5mM,Fe,Fe(NO)3・6H2O,0.5mM)、ii)ニッケルと鉄(Ni,NiCl2・6H2O,0.5mM,Fe,Fe(NO)3・6H2O,0.5mM)、iii) 亜鉛とマンガン(Zn,ZnSO4・7H2O,0.5mM,Mn,MnCl2・4H2O,0.5mM) 、iv) 亜鉛と鉄(Zn,ZnSO4・7H2O,0.5mM,Fe,Fe(NO)3・6H2O,0.5mM)イオンを添加して12時間の間、追加で培養した。 全体培養液を4℃、3500rpmで15分間、遠心分離した後上層液を捨てて大腸菌ペレットを集めた。PBSバッファーで大腸菌を洗浄する過程を三回繰り返した後、凍結乾燥機で真空状態で一日以上乾燥させた。ナノ粒子が合成されたことを透過電子顕微鏡を利用して調査した。
実施例3−1で製造された複合金属ナノ粒子の表面官能基を確認しようと、常温で赤外線分光器(Fourier−Transform Infrared Spectrophotometer,FT−IR)(Nicolet(登録商標)iS(登録商標)50,Thermo Scientific,USA)を利用して400〜4000cm−1の範囲で合成された複合金属ナノ粒子を分析した。
実施例3−1で製造されたナノ粒子の磁性特性を確認しようと、SQUID−VSM機能を備えたMPMS3(磁気測定システム)装備(SQUID−VSM)(Quantum Design,USA)を利用して磁気履歴曲線(M−H curve)を確認した。 磁気履歴曲線の測定は300K、−50K Oe〜+50K Oeで測定された。
4−1:組換え微生物を利用した複合金属ナノ粒子の合成
実施例1で製造された組換え微生物でメタロチオネインとファイトケラチン合成酵素の発現をIPTGで誘導して1時間後に、1時間後に大腸菌が培養されているLB液体培地(Tryptone 10g/L,Yeast extract 5g/L,NaCl 5g/L,pH 6.5)に銀(Ag,AgNO3,0.25,1mM)及びテルル(Te,NaTeO3,0.25,1mM)イオンを添加して12時間の間、追加で培養した。全体培養液を4℃、3500rpmで15分間、遠心分離した後上層液を捨てて大腸菌ペレットを集めた。PBSバッファーで大腸菌を洗浄する過程を三回繰り返した後、凍結乾燥機で真空状態で一日以上乾燥させた。ナノ粒子が合成されたことを透過電子顕微鏡を利用して調査した。
実施例4−1で製造された亜テルル酸銀(Ag2TeO3)ナノ粒子の表面官能基を確認しようと、常温で赤外線分光器(Fourier−Transform Infrared Spectrophotometer,FT−IR)(Nicolet(登録商標)iS(登録商標)50,Thermo Scientific,USA)を利用して400〜4000cm−1の範囲で亜テルル酸銀(Ag2TeO3)を分析した。
実施例4−1で製造された亜テルル酸銀(Ag2TeO3)ナノ粒子の磁性特性を確認しようと、SQUID−VSM機能を備えたMPMS3(磁気測定システム)装備(SQUID−VSM)(Quantum Design,USA)を利用して磁気履歴曲線(M−H curve)を確認した。磁気履歴曲線の測定は300K温度、−70K Oe〜+70K Oe 範囲の磁場の強さで行われた。
実施例4−1で製造された亜テルル酸銀(Ag2TeO3)ナノ粒子の電気化学的特性を確認しようと、電気化学分析装置(Potentiostat; Princeton applied research,VSP)を利用した。ナノ粒子の電気化学的特性は1M NaOH水溶液内で循環ボルタンメトリー(Cyclic voltammetry,CV)を利用して測定した。このために製造された亜テルル酸銀(Ag2TeO3)ナノ粒子をガラス炭素電極(Glassycarbon electrode)の上に乾燥させて作業電極にして、対電極としては白金線(Pt wire)を、基準電極としては銀/塩化銀(Ag/AgCl)を使用した。循環ボルタンメトリーの測定は‐0.4〜+0.5Vの電圧領域で10、20、30、50、100 mV/Sの異なる走査速度で測定した。
5−1:組換え微生物を利用した金属硫化物ナノ粒子の合成
実施例1で製造された組換え微生物でメタロチオネインとファイトケラチン合成酵素の発現をIPTGで誘導して1時間後に、1時間後に大腸菌が培養されているLB液体培地(Tryptone 10g/L,Yeast extract 5g/L,NaCl 5g/L,pH 6.5)にそれぞれ銀(Ag,AgNO3,1mM)、インジウム(In,InCl2・4H2O,1mM)、マンガン(Mn,MnSO4・5H2O ,1mM)またはすず(Sn,SnCl2・4H2O ,1mM)とともに硫(S,Na2S,1mM)を添加して12時間の間、追加で培養した。
実施例5−1で製造された金属硫化物ナノ粒子の表面官能基を確認しようと、常温で赤外線分光器(Fourier−Transform Infrared Spectrophotometer,FT−IR)(Nicolet(登録商標)iS(登録商標)50,Thermo Scientific,USA)を利用して400〜4000cm−1の範囲で合成された金属化合物のナノ粒子を分析した。
一部金属の場合、実施例1で製造された組換え微生物を培養する培地に金属イオンを添加しても金属ナノ粒子が合成されないことが示された。つまり、実施例1で製造された組換え大腸菌でメタロチオネインとファイトケラチン合成酵素の発現をIPTGで誘導して1時間後に大腸菌が培養されているLB液体培地(Tryptone 10g/L,Yeast extract 5g/L,NaCl 5g/L,pH 6.5)にそれぞれコバルト(Co,CoCl2・6H2O,0.5mM)、ニッケル(Ni,NiCl2・6H2O,0.5mM)、亜鉛(Zn,ZnSO4・7H2O,0.5mM)またはカドミウム(Cd,CdCl2,0.5mM)イオンを添加して12時間の間、追加で培養した。全体培養液を4℃、3500rpmで15分間、遠心分離した後上層液を捨てて大腸菌ペレットを集めた。PBSバッファーで大腸菌を洗浄する過程を三回繰り返した後、凍結乾燥機で真空状態で一日以上乾燥させた。本過程を通じて、ナノ粒子の合成の可否をを透過電子顕微鏡(Transmission electron microscopy,TEM)(Tecnai F20,Philips,Netherlands)を通じて調査したが、合成されるナノ粒子の形態を観察することができなくて、組換え大腸菌の形態だけが確認された(図21a,c,e,g)。
実施例1で製造された組換え微生物でメタロチオネインとファイトケラチン合成酵素の発現をIPTGで誘導して1時間後に大腸菌が培養されているLB液体培地(Tryptone 10g/L,Yeast extract 5g/L,NaCl 5g/L,pH 6.5)にランタン(La,La(NO3)3・6H2O,0.5mM)、セリウム(Ce、Ce(NO3)3・6H2O,0.5mM)、プラセオジム(Pr,Pr(NO3)3・6H2O,0.5mM)、ネオジウム(Nd,Nd(NO3)3・6H2O,0.5mM)、サマリウム(Sm,Sm(NO3)3・6H2O,0.5mM)、ユーロピウム(Eu,Eu(NO3)3・6H2O,0.5mM)またはガドリニウム(Gd,Gd(NO3)3・6H2O,0.5mM)イオンを添加して12時間の間、追加で培養した。全体培養液を4℃、3500rpmで15分間、遠心分離した後上層液を捨てて大腸菌ペレットを集めた。PBSバッファーで大腸菌を洗浄する過程を三回繰り返した後、凍結乾燥機で真空状態で一日以上乾燥させた。本過程を通じて、ナノ粒子の合成の可否をを透過電子顕微鏡(Transmission electron microscopy,TEM)(Tecnai F20,Philips,Netherlands)を通じて調査したが、合成されるナノ粒子の形態はすべて非晶質であることが確認され、結晶質ナノ粒子は合成されないことで示された(図23 a,c,e,g,i,k,m)。
Claims (20)
- 次の段階を含む単一の金属ナノ粒子の製造方法:
(a)メタロチオネインをコードする遺伝子及びファイトケラチン合成酵素をコードする遺伝子が導入された組換え微生物を培養する段階:
(b)亜鉛(Zn)、セレニウム(Se)、テルル(Te)、セシウム(Cs)、銅(Cu)、鉛(Pb)、ニッケル(Ni)、マンガン(Mn)、水銀(Hg)、コバルト(Co),クロム(Cr)、カドミウム(Cd)、ストロンチウム(Sr)、鉄(Fe)、金(Au)、銀(Ag)、プラセオジム(Pr)、ガドリニウム(Gd)、バリウム(Ba)、ジルコニウム(Zr)、モリブデン(Mo)、インジウム(In)、すず(Sn)、ランタン(La)、セリウム(Ce)、ネオジウム(Nd)、サマリウム(Sm)、イットリウム(Y)、アルミニウム(Al)及びユーロピウム(Eu)で構成された群から選択される金属イオンを添加した後、さらに培養して単一金属ナノ粒子を生成させる段階;及び
(c)前記生成された単一金属ナノ粒子を回収する段階。 - 前記微生物はバクテリア、酵母、藻類、古細菌及びカビで構成された群から選択されることを特徴とする、請求項1に記載の単一の金属ナノ粒子の製造方法。
- 前記メタロチオネインをコードする遺伝子は配列番号1で表示されるものであり、前記ファイトケラチン合成酵素をコードする遺伝子は配列番号2で表示されるものであることを特徴とする、請求項1に記載の単一の金属ナノ粒子の製造方法。
- 前記(b)段階の培地のpHは、前記組換え微生物を追加で培養して単一金属ナノ粒子を生成する最適のpHである8.0〜9.0に到達するように調整されることを特徴とする、請求項1に記載の単一の金属ナノ粒子の製造方法。
- 前記(b)段階の培地は、培養前の初期pHが7.3〜7.7に調節されることを特徴とする、請求項4に記載の単一の金属ナノ粒子の製造方法。
- 前記(a)段階の培地は、培養前の初期pHが6〜7であることを特徴とする、請求項5に記載の単一の金属ナノ粒子の製造方法。
- 前記生成された単一金属ナノ粒子は結晶質であることを特徴とする、請求項1に記載の単一の金属ナノ粒子の製造方法。
- 前記生成された 金属ナノ粒子は酸化コバルト(Co3O4)、水酸化ニッケル(Ni(OH)2)、過酸化亜鉛(ZnO)、水酸化カドミウム(Cd(OH)2)、水酸化コバルト(Co(OH)2)、炭酸バリウム(BaCO3)、水酸化ランタン(La(OH)3)、酸化セリウム(CeO2)、水酸化プラセオジム(Pr(OH)3)、水酸化ネオジウム(Nd(OH)3)、水酸化サマリウム(Sm(OH)3)、水酸化ユーロピウム(Eu(OH)3)、水酸化セリウム(Ce(OH)3)、水酸化イットリウム(Y(OH)3)、水酸化アルミニウム(Al(OH)3)及び水酸化ガドリニウム(Gd(OH)3))で構成された群から選択されることを特徴とする、請求項1に記載の単一の金属ナノ粒子の製造方法。
- 前記生成された金属ナノ粒子は酸化コバルト(Co3O4)、水酸化ニッケル(Ni(OH)2)、過酸化亜鉛(ZnO)及び水酸化カドミウム(Cd(OH)2)で構成された群から選択されることを特徴とする、請求項4に記載の単一の金属ナノ粒子の製造方法。
- 前記生成された金属ナノ粒子は水酸化ランタン(La(OH)3)、酸化セリウム(CeO2)、水酸化プラセオジム(Pr(OH)3)、水酸化ネオジウム(Nd(OH)3)、水酸化サマリウム(Sm(OH)3)、水酸化ユーロピウム(Eu(OH)3)及び水酸化ガドリニウム(Gd(OH)3))で構成された群から選択されることを特徴とする、請求項7に記載の単一の金属ナノ粒子の製造方法。
- 次の段階を含む複合金属ナノ粒子の製造方法:
(a)メタロチオネインをコードする遺伝子及びファイトケラチン合成酵素をコードする遺伝子が導入された組換え微生物を培養する段階:
(b)亜鉛(Zn)、セレニウム(Se)、テルル(Te)、セシウム(Cs)、銅(Cu)、鉛(Pb)、ニッケル(Ni)、マンガン(Mn)、水銀(Hg)、コバルト(Co),クロム(Cr)、カドミウム(Cd)、ストロンチウム(Sr)、鉄(Fe)、金(Au)、銀(Ag)、プラセオジム(Pr)、ガドリニウム(Gd)、バリウム(Ba)、ジルコニウム(Zr)、モリブデン(Mo)、インジウム(In)、すず(Sn)、ランタン(La)、セリウム(Ce)、ネオジウム(Nd)、サマリウム(Sm)、イットリウム(Y)、アルミニウム(Al)及びユーロピウム(Eu)で構成された群から選択される2つ以上の金属イオンを添加した後、さらに培養して複合金属ナノ粒子を生成させる段階;及び
(c)上記生成された複合金属ナノ粒子を回収する段階。 - 前記微生物はバクテリア、酵母、藻類、古細菌及びカビで構成された群から選択されることを特徴とする、請求項11に記載の複合金属ナノ粒子の製造方法。
- 前記メタロチオネインをコードする遺伝子は配列番号1で表示されるものであり、前記ファイトケラチン合成酵素をコードする遺伝子は配列番号2で表示されるものであることを特徴とする、請求項11に記載の 複合金属ナノ粒子の製造方法。
- 前記生成された複合金属ナノ粒子は酸化マンガン亜鉛 (ZnMn2O4)、酸化鉄亜鉛(ZnFe2O4)、酸化ニッケル鉄(NiFe2O4)、酸化コバルト鉄(CoFe2O4)、セレン化カドミウム(CdSe)、亜鉛カドミウム(CdZn)、テルル化カドミウム(CdTe)、亜鉛セレニド(SeZn)、カドミウムセレニド亜鉛(CdSeZn)、ストロンチウムガドリニウムス(SrGd)、プラセオジムガドリニウム(PrGd)、カドミウムセシウム(CdCs)、コバルト鉄(FeCo)、コバルト鉄ニッケル化合物(FeCoNi)及び亜テルル酸銀(Ag2TeO3)で構成される群から選択されることを特徴とする、請求項11に記載の 複合金属ナノ粒子の製造方法。
- 請求項11に記載の方法で製造された亜テルル酸銀(Ag2TeO3)のナノ粒子を含む造影剤。
- 請求項11に記載の方法で製造された亜テルル酸銀(Ag2TeO3)のナノ粒子を含む電極。
- 次の段階を含む金属硫化物のナノ粒子の製造方法:
(a)メタロチオネインをコードする遺伝子及びファイトケラチン合成酵素をコードする遺伝子が導入された組換え微生物を培養する段階:
(b)i)亜鉛(Zn)、セレニウム(Se)、テルル(Te)、セシウム(Cs)、銅(Cu)、鉛(Pb)、ニッケル(Ni)、マンガン(Mn)、水銀(Hg)、コバルト(Co),クロム(Cr)、カドミウム(Cd)、ストロンチウム(Sr)、鉄(Fe)、金(Au)、銀(Ag)、プラセオジム(Pr)、ガドリニウム(Gd)、バリウム(Ba)、ジルコニウム(Zr)、モリブデン(Mo)、インジウム(In)、すず(Sn)、ランタン(La)、セリウム(Ce)、ネオジウム(Nd)、サマリウム(Sm)及びユーロピウム(Eu)で構成された群から選択される金属イオン;及びii)硫(S)を添加した後、さらに培養して金属硫化物のナノ粒子を生成させる段階;及び
(c)前記生成された金属硫化物のナノ粒子を回収する段階。 - 前記微生物はバクテリア、酵母、藻類、古細菌及びカビで構成された群から選択されることを特徴とする、請求項17に記載の金属硫化物のナノ粒子の製造方法。
- 前記メタロチオネインをコードする遺伝子は配列番号1で表示されるものであり、前記ファイトケラチン合成酵素をコードする遺伝子は配列番号2で表示されるものであることを特徴とする、請求項17に記載の金属硫化物のナノ粒子の製造方法。
- 前記生成された金属硫化物のナノ粒子は、硫化銀(Ag2S)、硫化インジウム(InS)、硫化マンガン(MnS)、硫化亜鉛(ZnS)、硫化銅(CuS)、硫化カドミウム(CdS)、 硫化金(AuS)、硫化ニッケル(NiS)、硫化コバルト((CoS)、硫化水銀(HgS)及び硫化すず(SnS)で構成された群から選択されることを特徴とする、請求項17に記載の金属硫化物のナノ粒子の製造方法。
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