Bae W, Chen W, Mulchandani A, Mehra R K
Department of Chemical and Environmental Engineering, University of California, Riverside CA 92521, USA.
Biotechnol Bioeng. 2000 Dec 5;70(5):518-24. doi: 10.1002/1097-0290(20001205)70:5<518::aid-bit6>3.0.co;2-5.
A novel strategy using synthetic phytochelatins is described for the purpose of developing microbial agents for enhanced bioaccumulation of toxic metals. Synthetic genes encoding for several metal-chelating phytochelatin analogs (Glu-Cys)(n)Gly (EC8 (n = 8), EC11 (n = 11), and EC20 (n = 20)) were synthesized, linked to a lpp-ompA fusion gene, and displayed on the surface of E. coli. For comparison, EC20 was also expressed periplasmically as a fusion with the maltose-binding protein (MBP-EC20). Purified MBP-EC20 was shown to accumulate more Cd(2+) per peptide than typical mammalian metallothioneins with a stoichiometry of 10 Cd(2+)/peptide. Cells displaying synthetic phytochelatins exhibited chain-length dependent increase in metal accumulation. For example, 18 nmoles of Cd(2+)/mg dry cells were accumulated by cells displaying EC8, whereas cells exhibiting EC20 accumulated a maximum of 60 nmoles of Cd(2+)/mg dry cells. Moreover, cells with surface-expressed EC20 accumulated twice the amount of Cd(2+) as cells expressing EC20 periplasmically. The ability to genetically engineer ECs with precisely defined chain length could provide an attractive strategy for developing high-affinity bioadsorbents suitable for heavy metal removal.
本文描述了一种使用合成植物螯合肽的新策略，旨在开发用于增强有毒金属生物累积的微生物制剂。合成了编码几种金属螯合植物螯合肽类似物（Glu-Cys）(n)Gly（EC8（n = 8）、EC11（n = 11）和EC20（n = 20））的基因，将其与lpp-ompA融合基因连接，并展示在大肠杆菌表面。作为对照，EC20也作为与麦芽糖结合蛋白的融合蛋白在周质中表达（MBP-EC20）。结果表明，纯化的MBP-EC20每个肽积累的Cd(2+)比典型的哺乳动物金属硫蛋白更多，化学计量比为10个Cd(2+)/肽。展示合成植物螯合肽的细胞表现出金属积累的链长依赖性增加。例如，展示EC8的细胞积累了18 nmol Cd(2+)/mg干细胞，而展示EC20的细胞最多积累了60 nmol Cd(2+)/mg干细胞。此外，表面表达EC20的细胞积累的Cd(2+)量是周质表达EC20的细胞的两倍。通过基因工程精确控制EC链长的能力，可为开发适用于重金属去除的高亲和力生物吸附剂提供一种有吸引力的策略。
