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ISOLATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE ACTIVITY FROM SPIRODELA OLIGORHIZA
Citation:
Hoehamer, C. F., C S. Mazur, AND N L. Wolfe. ISOLATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE ACTIVITY FROM SPIRODELA OLIGORHIZA. Presented at Sixth International Symposium on Environmental Biotechnology and Fourth International Symposium on Cleaner Bioprocesses and Sustainable Development, Vera Cruz, Mexico, June 9-12, 2002.
Impact/Purpose:
Elucidate and model the underlying processes (physical, chemical, enzymatic, biological, and geochemical) that describe the species-specific transformation and transport of organic contaminants and nutrients in environmental and biological systems. Develop and integrate chemical behavior parameterization models (e.g., SPARC), chemical-process models, and ecosystem-characterization models into reactive-transport models.
Description:
An acid phosphatase activity from the aquatic plant Spirodela oligorhiza (duckweed) was isolated and partially characterized. S. oligorhiza was grown in a hydroponic growth medium, harvested, and ground up in liquid nitrogen. The ground plant material was added to a biological buffer, and after ultrafiltration and differential centrifugation, two cell-free enzyme fractions were prepared, the soluble and membrane. The soluble fraction of enzymes displayed low amounts of inducible acid phosphatase activity, while it was not detected in the membrane fraction. This enzyme activity was further isolated from the extract by salt precipitation and fast pressure liquid chromatography using columns containing Phenyl-sepharose and Resource S cation-exchange matrices. Acid phosphatase activity in the plant extracts was monitored colormetrically by measuring the hydrolysis of -nitrophenol from the organophosphate substrates, methyl paraoxon and -nitrophenolphosphate. The acid phosphatase activity was determined to have a pH optimum of 6.0 at 25 C, and no other cofactors were required for enzyme activity. Zymogram staining techniques using 4-methylumbelliferyl phosphate visualized two acid phosphatase isoforms on nondenatured semi-native SDS-PAGE gels which corresponded to protein bands on gels stained with Coomassie brilliant blue. While only minor contaminants were observed on the protein gels after the cation exchange step, this enzyme(s) will soon be purified to homogeneity and further characterized with respect to its chemical and physical properties. Additionally, a variety of other organophosphate compounds remain to be tested as substrates with this phosphatase to determine not only its substrate specificity, but the overall potential of this plant in pesticide remediation.