Perchlorate Removal in groundwater by perchlorate reductases from the perchlorate respiring bacterium, perc1ace
[Note: See PDF for correct symbols.] Perchlorate (ClO4-) is an important energetic component of solid rocket fuel. The major source of ClO4- pollution is the military, space program and supporting industries. ClO4- is recalcitrant in the environment and is potentially toxic. The California Department of Health Services adopted an action level of 4 ppb for perchlorate in potable water. Microorganisms that reduce ClO4- to chloride and molecular oxygen have been isolated. For designing an efficient biological-based ground water ClO4- remediation strategy, the biochemical and molecular data on the enzymatic reduction of ClO4- are needed. The ClO4- respiring organism, perc1ace when grown using either ClO4- or NO3- as a terminal electron acceptor produced ClO4- reductase to a significant extent. The ClO4- reductase activity appeared to be within the periplasmic space, with activities as high as 14, 000 nmol-1 min-1 mg protein-1, indicating that it is a soluble enzyme. A ClO4- reductase from cell-free extracts of perc1ace was purified 10-fold by ion-exchange and molecular exclusion fast protein liquid chromatography (FPLC). The ClO4- reductase catalyzed the reduction of ClO4- at a Vmax and Km of 4.8 Units mg protein-1 and 34.5 M, respectively. Maximal activity was recorded at 25-30oC and pH 7.5 – 8.0. Perc1ace ClO4- reductase is a dimer with molecular masses of 35.07 kDa and 75.1 kDa determined by SDS-PAGE. Matrix-Assisted Laser Desorption Ionization-Time of Flight/Mass Spectrometry (MALDI-TOF/MS) analysis of the 35 kDa protein revealed several tryptic peptides. To study the genetic determinants of ClO4- reductase, the amino terminal sequences of 22 tryptic peptides of the approximately 35 kDa ClO4- reductase subunit were obtained by electrospray mass spectrometry. GenBank Blast analysis of the amino acid sequences revealed similarity to reductases, dehydrogenases and heme proteins. In batch studies of in vitro reduction of perchlorate, perc1ace ClO4- reductase reduced perchlorate in water with either NADH or methyl viologen as an electron donor. Less enzyme activity was observed with methanol and ethanol. Experiments showed that ClO4- reductase immobilized to Ca alginate reduced ClO4-. Additional studies are focusing on optimization of reaction conditions for perchlorate reduction by immobilized perchlorate reductases, molecular characterization of the overall genetic determinants of ClO4- bioreduction by perc1ace by cloning the genes using degenerate primers designed from the amino acid sequences of ClO4- reductase tryptic peptides and over-expression of recombinant ClO4- reductase. Such a recombinant enzyme available in large quantities can be immobilized and safely used for the treatment of perchlorate contaminated ground water on site. Treatment systems designed to employ cell-free enzymes catalyze the ClO4- reduction reaction without the production of biomass wastes. Moreover, the spent enzymes can be regenerated and reused, substantially reducing cost.