Crystallizing extracellular protein reveals paths for silver mineralization and recovery

Heavy metal pollution calls for green recovery strategies. In response to metal stress, bacteria secret extracellular proteins (ECPs), but the specific role of individual ECP in silver (Ag2+) mineralization and recovery remains unexplored. Here, we investigated how a single ECP from silver-hypertolerant Enterobacter cloacae mediates Ag²⁺ mineralization. Proteomics (LC-MS/MS, MALDI-TOF) identified the 15.6 kDa protein as an inosine-monophosphate dehydrogenase (ImpD) homolog, whose secretion peaks in medium containing 15.9 mg L⁻¹ Ag²⁺. Partially purified ImpD crystallized at 20–30 °C. Time-resolved in-situ crystallography and X-ray diffraction captured monomers assembling into donut-shaped hexamers that weave into thread-like lattices; these ordered scaffolds template orientation-specific nucleation of Ag-rich crystals, enabling quantitative silver capture from solution. This previously unrecognized single-protein mediated biomineralization mechanism reveals ECPs as programmable bio-lixiviants, offering a low-energy, solvent-free route to selective metal recovery and expanding the toolkit for biometallurgy and environmental remediation.重金属污染亟需绿色回收修复策略。为应对金属胁迫，细菌会分泌胞外蛋白（extracellular proteins, ECPs），但单一胞外蛋白在银离子（Ag²⁺）矿化与回收过程中的具体作用仍未被探明。本研究针对一株高耐银的阴沟肠杆菌（Enterobacter cloacae）分泌的单一胞外蛋白如何介导银离子矿化展开探究。通过蛋白质组学（液相色谱-串联质谱LC-MS/MS、基质辅助激光解吸电离飞行时间质谱MALDI-TOF）分析，该15.6 kDa蛋白被鉴定为肌苷单磷酸脱氢酶（inosine-monophosphate dehydrogenase, ImpD）的同源蛋白，其分泌量在含15.9 mg·L⁻¹银离子的培养基中达到峰值。部分纯化后的ImpD可在20~30 ℃条件下结晶。时间分辨原位晶体学与X射线衍射技术观测到，单体组装成甜甜圈状六聚体，进而交织形成丝状晶格；这些有序支架可定向引导富银晶体的成核过程，实现溶液中银离子的定量捕获。这一此前未被报道的单一蛋白介导的生物矿化机制，证实胞外蛋白可作为可编程的生物浸出剂（bio-lixiviants），为选择性金属回收提供了低能耗、无溶剂的新路径，同时拓展了生物冶金与环境修复的工具库。