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金属ナノ粒子生成細菌による金属沈着機構の解明

加藤, 由悟 東京大学 DOI:10.15083/0002006869

2023.03.24

概要





















加藤

由悟

序論では研究の背景、目的を述べている。金属ナノ粒子は 1~100 nm の金
属の結晶であり、表面の影響が大きくなるだけではなく、全体を構成する原子核
と電子の数が有限となることで量子サイズ効果と呼ばれる独特の物性を持ち、
医療や工学の分野で数多く利用されている。微生物による金属ナノ粒子合成は、
エネルギーを必要としない新たな手法であるだけでなく、溶液中からの金属回
収手法としても注目を集めているが、そのメカニズムはほとんど明らかになっ
ていない。本論文は菌体内に金属をナノ粒子として生成する微生物に着目し、そ
の沈着機構の解明を目指して研究を行ったものである。
第一部では乳酸菌の金ナノ粒子合成における粒子径制御のメカニズムを追究
している。これまでの研究から乳酸菌の細胞膜に存在する糖脂質ジグリコシル
ジアシルグリセロール(DGDG)が金ナノ粒子の合成に関与することが明らか
になっているが、乳酸菌に含まれる成分には DGDG の他に金ナノ粒子の合成に
寄与している物質が存在することが示唆されているものの、どのような物質で
あるか同定されていなかった。クライオ電子顕微鏡により観察したところ、金ナ
ノ粒子は乳酸菌の細胞膜の表面近傍で合成されていることが示され、菌体外に
放出されている成分(菌体外成分)に粒子の凝集を抑え粒径を小さくする物質が
存在しているとの仮説を立て、菌体外成分を対象に探索した。アミドカラムを用
いた高速液体クロマトグラフィーにより分離した菌体外成分のうち、金ナノ粒
子合成の活性画分を核磁気共鳴スペクトルおよび質量分析装置を用いて分析し
た結果、アセチル化された三糖 lacto-N-triose(LNtri)と乳酸が金ナノ粒子の
合成活性を持つことを示した。特定した物質を合わせて用いることで金ナノ粒
子の合成に成功したことから、DGDG を含めた3つの物質が協調することで乳
酸菌により金ナノ粒子が生成されるメカニズムを初めて明らかにした。本研究
成果による乳酸菌の金ナノ粒子生成メカニズムの解明は、微生物を用いた金属
ナノ粒子合成の効率化や生体を模倣した新たな金属ナノ粒子合成手法の開発に
繋がると期待される。
第二部では金属濃度の高い水域から鉛ナノ粒子合成能を持つ環境微生物を単

離し、その金属濃集のメカニズムの解明を行うことを目的に研究を行っている。
その結果、尾小屋鉱山周辺の流域から鉛ナノ粒子を菌体周辺に合成する
Pseudomonas 属 KKY-29 株を分離した。TEM による観察や X 線回折装置を用
いた分析から KKY-29 株は酢酸鉛水溶液中でインキュベートすることで緑鉛鉱
(Pb5(PO4)3Cl) および水白鉛鉱 (2PbCO3・Pb(OH)2) のナノ粒子を菌体内に合成
していることが分かった。リン酸代謝に関する遺伝子の発現解析の結果、KKY29 株がポリリン酸を分解することで鉛の結晶化を行っていることが示唆された。
次に、KKY-29 株に含まれる分散剤の特定を目指した実験を行っている。KKY29 の超音波破砕により得られた抽出液、およびその残渣をろ過することで得ら
れたろ液を分析に供している。超音波破砕抽出液およびその残渣をろ過した液
の両方の鉛濃度を測定したところ、両方に鉛ナノ粒子が存在しているものと考
え、両方ともにゲルろ過カラムを用いた HPLC-ICP-MS(高速液体クロマトグ
ラフィー誘導結合プラズマ質量分析)により鉛ナノ粒子の存在する画分を分取
し、ポリアクリルアミドゲル電気泳動に供し、分離を行った。バンドパターンか
ら得られた鉛結合物質と思われるバンドを LC-MS/MS(液体クロマトグラフィ
ー質量分析計)に供したところ、外膜タンパク質として金属イオン輸送に寄与す
る TonB-dependent receptor(TBDR)が同定された。KKY-29 株の TBDR をコ
ードする遺伝子の発現解析の結果、鉛イオン添加時に発現量が増加しているこ
とが分かり、TBDR が鉛ナノ粒子の沈着に寄与していると考えられた。また、
ろ液からは菌体内に存在する酵素である Arylsulfatase や Tyrosinase が同定さ
れ、これらが鉛ナノ粒子の合成に寄与している可能性が示唆された。このことか
ら KKY-29 株では、細胞内に取り込まれた鉛イオンが、菌体内で分解されたリ
ン酸イオンと結合して結晶になると考えられた。これらの結果から、細胞内のタ
ンパク質が配位することによって結晶成長が抑えられ、ナノ粒子として存在す
ると提案している。
これらの研究成果は、学術上応用上寄与するところが少なくない。よって、
審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。

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