A Physiological Study on Microbiological Silver Recovery Process

Research Project

Project/Area Number	62550720
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	発酵工学
Research Institution	Shimane University
Principal Investigator	KOIZUMI Jun-ichi Shimane University, Associate Professor, 農学部, 助教授 (00150334)
Project Period (FY)	1987 – 1988
Project Status	Completed (Fiscal Year 1988)
Budget Amount *help	¥1,900,000 (Direct Cost: ¥1,900,000) Fiscal Year 1988: ¥600,000 (Direct Cost: ¥600,000) Fiscal Year 1987: ¥1,300,000 (Direct Cost: ¥1,300,000)
Keywords	Ag-resistance determinant / Bioaccumulation of silver / Plasmid / クローニング / 銀耐性 / 金属包接短鎖ペプチド
Research Abstract	An isolate AGR7, silver-resistant bacterium, was identified as Enterobacter cloacae through physiological characterization. A large plasmid pAGR7 was detected from its lysozyme lysate, of which molecular weight was more than 20 Mdal. The plasmid pAGR7 prepared from En. cloacae AGR was capable of transforming Escherichia coli C600-1. The magnitude of silver resistance in E. coli C600-1 was enhanced more than 80 fold from <20 mg/l to 1,600 mg/l through the transformation. The transformant obtained also silver-accumulation ability. Using a ligation mixture of pUC18 and DNA fragments from En. cloacae AGR , E. coli JM109 was transformed. Sixty six of ca, 15,000 transformant colonies could grow on L agar plate containing 200 mg/l of silver ion. None of 66 transformants, however, could grow in L broth containing 40 mg/l of silver ion. It was concluded that the expression of silver-resistance determinant cloned into pUC18 differed from that in the intact pAGR7 in efficiency. Cytoplasmic fractions were recovered from the lysares of En. cloacae AGR, which was inculbated in various conditions and disrupted at 10,000 psi with French Press. The fraction were, further, fractionated with a high performance liquid chromatography with a gel filtration column. The amounts of peptides and nucleic acids in the elution were monitored by UV 280 nm and 254 nm, respectively. The concentration of silver in the elution was measured with either an inductively coupled plasma or an atomic absorption detector. In the cytoplasm undergoing silver dosage for 2 h, the increase of RNA was observable, and the induction nucleic acidsynthesis was confirmed. The induction, however, deteriorated the ability of silver accumulation, which was investigated separately. A short peptide was detected, of which molecular weight was ca. 7,000 and mercapto groups were rich; it resembled metallothionein existing in eukaryote in these characteristics.